U.S. patent number 7,083,793 [Application Number 10/107,857] was granted by the patent office on 2006-08-01 for tango 243 polypeptides and uses thereof.
This patent grant is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Christopher C. Fraser.
United States Patent |
7,083,793 |
Fraser |
August 1, 2006 |
Tango 243 polypeptides and uses thereof
Abstract
The invention provides isolated nucleic acid molecules,
designated TANGO 228 nucleic acid molecules, which encode secreted
proteins with homology to the rat MCA-32 protein, isolated nucleic
acid molecules, designated TANGO 240 nucleic acid molecules, which
encode secreted proteins with homology to the Mycobacterium
tuberculosis hypothetical protein Rv0712, and isolated nucleic acid
molecules, designated TANGO 243 nucleic acid molecules, which
encode proteins with homology to human PLAP (phospholipase
A2-activating protein). The invention also provides antisense
nucleic acid molecules, expression vectors containing the nucleic
acid molecules of the invention, host cells into which the
expression vectors have been introduced, and non-human transgenic
animals in which a nucleic acid molecule of the invention has been
introduced or disrupted. The invention still further provides
isolated polypeptides, fusion polypeptides, antigenic peptides and
antibodies. Diagnostic, screening and therapeutic methods utilizing
compositions of the invention are also described/disclosed.
Inventors: |
Fraser; Christopher C.
(Lexington, MA) |
Assignee: |
Millennium Pharmaceuticals,
Inc. (Cambridge, MA)
|
Family
ID: |
46280435 |
Appl.
No.: |
10/107,857 |
Filed: |
March 26, 2002 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20030170784 A1 |
Sep 11, 2003 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
09928788 |
Aug 13, 2001 |
|
|
|
|
09514009 |
Feb 25, 2000 |
|
|
|
|
09259387 |
Feb 26, 1999 |
|
|
|
|
Current U.S.
Class: |
424/192.1;
530/350; 536/23.2; 536/23.4 |
Current CPC
Class: |
C07K
14/4705 (20130101); C07K 14/52 (20130101); C07K
14/47 (20130101); C07K 14/705 (20130101); C07K
16/18 (20130101); C07K 16/28 (20130101); C12Q
1/025 (20130101); C12Q 1/6813 (20130101); C12Q
1/686 (20130101); G01N 33/53 (20130101); G01N
33/68 (20130101); C07H 21/04 (20130101); C07K
14/475 (20130101); C07K 16/22 (20130101); G01N
33/6872 (20130101); A61K 38/00 (20130101); A61K
39/00 (20130101); C12N 15/86 (20130101); C12N
2800/10 (20130101); Y10T 436/143333 (20150115) |
Current International
Class: |
A61K
39/00 (20060101); C07K 19/00 (20060101); C07K
14/435 (20060101) |
Field of
Search: |
;530/350 ;536/23.5 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO 91/10683 |
|
Jul 1991 |
|
WO |
|
WO 94/01548 |
|
Jan 1994 |
|
WO |
|
WO 98/56400 |
|
Dec 1998 |
|
WO |
|
WO 99/33982 |
|
Jul 1999 |
|
WO |
|
Other References
Wang et al., Database GenEmbl, Accession No. U17901, Oct. 22, 1995.
cited by examiner .
Janknecht et al., PNAS (USA), Viol. 88, pp. 8972-8976, Oct. 1991.
cited by examiner .
Vukicevic et al., 1996, PNAS USA 93:9021-9026. cited by examiner
.
U.S. Appl. No. 60/049,316. cited by examiner .
Cross et al., "Purification of CpG islands using a methylated DNA
binding column" Nature Genetics 6:236-244, Mar. 1994. cited by
other .
MacDonald et al., Accession No. Z62057, Oct. 1995. cited by other
.
Birren et al., Accession No. AC009994, Dec. 7, 1999. cited by other
.
Chopra et al., Accession No. AF083395, Mar., 4, 1999. cited by
other .
Cole et al., Accession No. Z84395, Jun. 17, 1998. cited by other
.
Pirozzi et al., Accession No. U39546, Dec. 28, 1995. cited by other
.
Peterson, J. W., et al., "Cholera Toxin Induces Synthesis of
Phospholipase A.sub.2-Activating Protein," Infect. Immun.,
64(6):2137-2143 (1996). cited by other .
Chopra, A. K., et al., "Molecular Characterization of cDNA for
Phospholipase A.sub.2-Activating Protein," Biochim. Biophys. Acta,
1444:125-130 (1999). cited by other .
Bomalaski, J. S., et al., "Phospholipase A.sub.2-Activating Protein
Induces the Synthesis of IL-1 and TNF in Human Monocytes," J.
Immunol., 154(8):4027-4031 (1995). cited by other .
Wang, H., et al., "Cloning of a Rat cDNA Encoding a Protein with
High Homology to Mouse Phospholipase A2-Activating Protein," Gene,
161:237-241 (1995). cited by other.
|
Primary Examiner: O'Hara; Eileen B.
Attorney, Agent or Firm: Millennium Pharmaceuticals,
Inc.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
09/928,788, filed Aug. 13, 2001, now abandoned, which is a
continuation of U.S. application Ser. No. 09/514,009, filed Feb.
25, 2000, now abandoned, which is a continuation-in-part of U.S.
application Ser. No. 09/259,387, filed Feb. 26, 1999, now
abandoned.
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence that has at
least 95% amino acid sequence identity with SEQ ID NO:21, wherein
the polypeptide is capable of modulating phospholipase A enzyme
activation; b) a polypeptide comprising the amino acid sequence of
a fragment of SEQ ID NO:21 comprising at least 740 contiguous amino
acids, wherein the polypeptide is capable of modulating
phospholipase A enzyme activation; c) a polypeptide which has an
amino acid sequence that is encoded by a nucleic acid molecule
comprising a nucleotide sequence which has at least 92% nucleotide
sequence identity with the nucleotide sequence of SEQ ID NO:19,
wherein the polypeptide is capable of modulating phospholipase A
enzyme activation; d) a polypeptide encoded by a nucleic acid
molecule comprising a nucleotide sequence which has at least 93%
nucleotide sequence identity with the nucleotide sequence of SEQ ID
NO:20, wherein the polypeptide is capable of modulating
phospholipase A enzyme activation; e) a polypeptide comprising an
amino acid sequence that has at least 95% amino acid sequence
identity with the amino acid sequence encoded by the cDNA insert of
the plasmid encoding TANGO 243 deposited with the ATCC under
Accession Number 207116, wherein the polypeptide is capable of
modulating phospholipase A enzyme activation; and f) a polypeptide
comprising an amino acid sequence encoded by a fragment of the cDNA
insert of the plasmid encoding TANGO 243 deposited with the ATCC
under Accession Number 207116, wherein the fragment encodes at
least 740 contiguous amino acids and wherein the polypeptide is
capable of modulating phospholipase A enzyme activation; wherein
percent nucleotide sequence identity and percent amino acid
sequence identity are determined over the full length of SEQ ID
NO:19, 20 or 21 or over the full length of the cDNA insert of the
plasmid encoding TANGO 243 deposited with the ATCC under Accession
Number 207116 using the algorithm of Myers and Miller (CABIOS 4:11
17 (1988)).
2. The isolated polypeptide of claim 1, wherein said polypeptide
comprises the amino acid sequence of SEQ ID NO:21 or a fragment of
SEQ ID NO:21 comprising at least 740 contiguous amino acids.
3. The polypeptide of claim 1 further comprising a heterologous
amino acid sequence.
4. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID
NO:45; and b) a polypeptide comprising an amino acid sequence which
has at least 95% amino acid sequence identity with SEQ ID NO:45,
wherein the polypeptide is capable of modulating phospholipase A
enzyme activation and wherein amino acid sequence identity is
determined over the full length of SEQ ID NO:45 using the algorithm
of Myers and Miller (CABIOS 4:11 17 (1988)).
5. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID
NO:21; b) a polypeptide comprising a fragment of the amino acid
sequence SEQ ID NO:21, wherein the fragment comprises at least 740
contiguous amino acids of SEQ ID NO:21 and wherein the polypeptide
is capable of modulating phospholipase A enzyme activation; c) a
polypeptide comprising an amino acid sequence encoded by the cDNA
insert of the plasmid encoding TANGO 243 deposited with the ATCC
under Accession Number 207116; and d) a polypeptide comprising an
amino acid sequence comprising at least 740 contiguous amino acids
encoded by the cDNA insert of the plasmid encoding TANGO 243
deposited with the ATCC under Accession Number 207116, wherein the
polypeptide is capable of modulating phospholipase A enzyme
activation.
6. An isolated polypeptide comprising an amino acid sequence that
has at least 95% amino acid sequence identity with SEQ ID NO:21,
wherein the polypeptide is capable of modulating phospholipase A
enzyme activation and wherein percent amino acid sequence identity
is determined over the full length of SEQ ID NO:21 using the
algorithm of Myers and Miller (CABIOS 4:11 17 (1988)).
7. An isolated polypeptide selected from the group consisting of:
a) a polypeptide which has an amino acid sequence that is encoded
by a nucleic acid molecule comprising a nucleotide sequence which
has at least 95% nucleotide sequence identity with the nucleotide
sequence of SEQ ID NO:19, wherein the polypeptide is capable of
modulating phospholipase A enzyme activation; and b) a polypeptide
encoded by a nucleic acid molecule comprising a nucleotide sequence
which has at least 95% nucleotide sequence identity with the
nucleotide sequence of SEQ ID NO:20, wherein the polypeptide is
capable of modulating phospholipase A enzyme activation; wherein
percent nucleotide sequence identity is determined over the full
length of SEQ ID NO:19 or 20 using the algorithm of Myers and
Miller (CABIOS 4:11 17 (1988)).
Description
BACKGROUND OF THE INVENTION
Many secreted proteins, for example, cytokines and cytokine
receptors, play a vital role in the regulation of cell growth, cell
differentiation, and a variety of specific cellular responses. A
number of medically useful proteins, including erythropoietin,
granulocyte-macrophage colony stimulating factor, human growth
hormone, and various interleukins, are secreted proteins. Thus, an
important goal in the design and development of new therapies is
the identification and characterization of secreted and
transmembrane proteins and the genes which encode them.
Many secreted proteins are receptors which bind a ligand and
transduce an intracellular signal, leading to a variety of cellular
responses. The identification and characterization of such a
receptor enables one to identify both the ligands which bind to the
receptor and the intracellular molecules and signal transduction
pathways associated with the receptor, permitting one to identify
or design modulators of receptor activity, e.g., receptor agonists
or antagonists and modulators of signal transduction.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery
of cDNA molecules which encode the TANGO 228, 240, and 243
proteins, all of which are either wholly secreted or transmembrane
proteins.
The TANGO 228 proteins are homologous to rat surface protein MCA-32
(mast cell Ag-32), a component of the immunologic pathway.
The TANGO 240 proteins are homologous to the Mycobacterium
tuberculosis conserved hypothetical protein Rv0712.
The TANGO 243 proteins share significant homology to human PLAP
(phospholipase A2-activating protein), a modulator of arthropathic
disorders.
The TANGO 228, TANGO 240, and TANGO 243 proteins, fragments,
derivatives, and variants thereof are collectively referred to
herein as "polypeptides of the invention" or "proteins of the
invention." Nucleic acid molecules encoding the polypeptides or
proteins of the invention are collectively referred to as "nucleic
acids of the invention."
The nucleic acids and polypeptides of the present invention are
useful as modulating agents in regulating a variety of cellular
processes. Accordingly, in one aspect, this invention provides
isolated nucleic acid molecules encoding a polypeptide of the
invention or a biologically active portion thereof. The present
invention also provides nucleic acid molecules which are suitable
for use as primers or hybridization probes for the detection of
nucleic acids encoding a polypeptide of the invention.
The invention features nucleic acid molecules which are at least
30% (or 35%, 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%)
identical to the nucleotide sequence of SEQ ID NO:1, the nucleotide
sequence of the cDNA insert of a clone deposited with ATCC as
Accession Number 207116, or a complement thereof.
The invention features nucleic acid molecules which are at least
48% (or 50%, 55%, 60%, 65%, 75%, 85%, 95%, or 98%) identical to the
nucleotide sequence of SEQ ID NO:2, the nucleotide sequence of the
cDNA insert of a clone deposited with ATCC as Accession Number
207116, or a complement thereof.
The invention features nucleic acid molecules which are at least
30% (or 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%) identical
to the nucleotide sequence of SEQ ID NO:13 or 14, the nucleotide
sequence of the cDNA insert of a clone deposited with ATCC as
Accession Number 207116, or a complement thereof.
The invention features nucleic acid molecules which are at least
80% (or 82%, 85%, 87%, 90%, 92%, 95%, or 98%) identical to the
nucleotide sequence of SEQ ID NO:19, the nucleotide sequence of the
cDNA insert of a clone deposited with ATCC as Accession Number
207116, or a complement thereof.
The invention features nucleic acid molecules which are at least
93% (or 94%, 95%, 96%, 97%, or 98%) identical to the nucleotide
sequence of SEQ ID NO:20, the nucleotide sequence of the cDNA
insert of a clone deposited with ATCC as Accession Number 207116,
or a complement thereof.
The invention features nucleic acid molecules which include a
fragment of at least 310 (400, 500, 600, 800, 1000, 1250, 1500,
1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, or
4020) nucleotides of the nucleotide sequence of SEQ ID NO:1 the
nucleotide sequence of the cDNA of ATCC Accession Number 207116, or
a complement thereof.
The invention features nucleic acid molecules which are at least
30% (or 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%) identical
to the nucleotide sequence of SEQ ID NO:31 or 32, or a complement
thereof.
The invention features nucleic acid molecules which are at least
30% (or 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%) identical
to the nucleotide sequence of SEQ ID NO:39 or 40, or a complement
thereof.
The invention features nucleic acid molecules which include a
fragment of at least 515 (530, 550, 600, 700, 800, 900, 1000, 1100,
1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or
2150) nucleotides of the nucleotide sequence of SEQ ID NO:13, the
nucleotide sequence of the cDNA of ATCC Accession Number 207116, or
a complement thereof.
The invention features nucleic acid molecules which include a
fragment of at least 2220 (2260, 2300, 2340, 2380, 2420, 2460,
2500, 2540, 2580, 2620, 2660, 2700, 2740, 2780, or 2800)
nucleotides of the nucleotide sequence of SEQ ID NO:19, the
nucleotide sequence of the cDNA of ATCC Accession Number 207116, or
a complement thereof.
The invention features nucleic acid molecules which include a
fragment of at least 50 (100, 150, 200, 250, 300, 350, 400, 450,
500, 550, 600, 650, 700, 750, 800, or 900) nucleotides of the
nucleotide sequence of SEQ ID NO:31 or 32, or a complement
thereof.
The invention features nucleic acid molecules which include a
fragment of at least 50 (100, 150, 200, 250, 300, 350, 400, 450
500, 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,
1600, 1700, 1800, 1900, 2000, 2100, or 220) nucleotides of the
nucleotide sequence of SEQ ID NO:39 or 40, or a complement
thereof.
The invention also features nucleic acid molecules which include a
nucleotide sequence encoding a protein having an amino acid
sequence that is at least 30% (or 35%, 40%, 45%, 50%, 55%, 60%,
65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of
SEQ ID NO:3, the amino acid sequence encoded by the cDNA of ATCC
Accession Number 207116, or a complement thereof.
The invention also features nucleic acid molecules which include a
nucleotide sequence encoding a protein having an amino acid
sequence that is at least 35% (or 40%, 45%, 50%, 55%, 65%, 75%,
85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID
NO:15, the amino acid sequence encoded by the cDNA of ATCC
Accession Number 207116, or a complement thereof.
The invention also features nucleic acid molecules which include a
nucleotide sequence encoding a protein having an amino acid
sequence that is at least 93% (or 94%, 95%, 96%, 97% or 98%)
identical to the amino acid sequence of SEQ ID NO:21, the amino
acid sequence encoded by the cDNA of ATCC Accession Number 207116,
or a complement thereof.
The invention also features nucleic acid molecules which include a
nucleotide sequence encoding a protein having an amino acid
sequence that is at least 35% (or 40%, 45%, 50%, 55%, 65%, 75%,
85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID
NO:33, the amino acid sequence encoded by the cDNA of ATCC
Accession Number 207116, or a complement thereof.
The invention also features nucleic acid molecules which include a
nucleotide sequence encoding a protein having an amino acid
sequence that is at least 35% (or 40%, 45%, 50%, 55%, 65%, 75%,
85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID
NO:41, the amino acid sequence encoded by the cDNA of ATCC
Accession Number 207116, or a complement thereof.
The invention also features nucleic acid molecules which include a
nucleotide sequence encoding a protein having an amino acid
sequence that is at least 35% (or 40%, 45%, 50%, 55%, 65%, 75%,
85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID
NO:45, the amino acid sequence encoded by the cDNA of ATCC
Accession Number 207116, or a complement thereof.
In preferred embodiments, the nucleic acid molecules have the
nucleotide sequence of SEQ ID NO:1, 2, 13, 14, 19, 20, 31, 32, 39,
40, 44 or the nucleotide sequence of the cDNA of ATCC Accession
Number 207116.
Also within the invention are nucleic acid molecules which encode a
fragment of a polypeptide having the amino acid sequence of SEQ ID
NO:3, or a fragment including at least 15 (25, 30, 50, 75, 100,
125, 150, 175, 200, 225, 250, 275, 300, or 340) contiguous amino
acids of SEQ ID NO:3, or the amino acid sequence encoded by the
cDNA of ATCC Accession Number 207116.
Also within the invention are nucleic acid molecules which encode a
fragment of a polypeptide having the amino acid sequence of SEQ ID
NO:15, or a fragment including at least 15 (25, 30, 50, 75, 100,
125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 360, or 370)
contiguous amino acids of SEQ ID NO:15, or the amino acid sequence
encoded by the cDNA of ATCC Accession Number 207116.
Also within the invention are nucleic acid molecules which encode a
fragment of a polypeptide having the amino acid sequence of SEQ ID
NO:21, or a fragment including at least 740 (745, 750, 755, 760,
765, 770, 775, 780, 785, or 790) contiguous amino acids of SEQ ID
NO:21, or the amino acid sequence encoded by the cDNA of ATCC
Accession Number 207116.
Also within the invention are nucleic acid molecules which encode a
fragment of a polypeptide having the amino acid sequence of SEQ ID
NO:33, or a fragment including at least 15 (25, 30, 50, 75, 100,
125, 150, 175, 200, 225, or 240) contiguous amino acids of SEQ ID
NO:33.
Also within the invention are nucleic acid molecules which encode a
fragment of a polypeptide having the amino acid sequence of SEQ ID
NO:41, or a fragment including at least 15 (25, 30, 50, 75, 100,
125, 150, 175, 200, 250, 300, 350 or 370) contiguous amino acids of
SEQ ID NO:41.
Also within the invention are nucleic acid molecules which encode a
fragment of a polypeptide having the amino acid sequence of SEQ ID
NO:21, or a fragment including at least 740 (745, 750, 755, 760,
765, 770, 775, 780, 785, 790, 800, 825, 850, or 880) contiguous
amino acids of SEQ ID NO:21, or the amino acid sequence encoded by
the cDNA of ATCC Accession Number 207116.
The invention includes nucleic acid molecules which encode a
naturally occurring allelic variant of a polypeptide comprising the
amino acid sequence of SEQ ID NO:3, 15, 21, 33, 41, or 45, or the
amino acid sequence encoded by the cDNA of ATCC Accession Number
207116, wherein the nucleic acid molecule hybridizes to a nucleic
acid molecule consisting of a nucleic acid sequence encoding SEQ ID
NO:3, 15, 21, 33, 41, or 45, the amino acid sequence encoded by the
cDNA of ATCC Accession Number 207116, or a complement thereof under
stringent conditions.
Also within the invention are isolated polypeptides or proteins
having an amino acid sequence that is at least about 30%,
preferably 35%, 45%, 55%, 65%, 75%, 85%, 95%, or 98% identical to
the amino acid sequence of SEQ ID NO:3, or the amino acid sequence
encoded by the cDNA of ATCC Accession Number 207116.
Also within the invention are isolated polypeptides or proteins
having an amino acid sequence that is at least about 35%,
preferably 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical
to the amino acid sequence of SEQ ID NO:15, or the amino acid
sequence encoded by the cDNA of ATCC Accession Number 207116.
Also within the invention are isolated polypeptides or proteins
having an amino acid sequence that is at least about 93%,
preferably 94%, 95%, 96%, 97% or 98% identical to the amino acid
sequence of SEQ ID NO:21, or the amino acid sequence encoded by the
cDNA of ATCC Accession Number 207116.
Also within the invention are isolated polypeptides or proteins
having an amino acid sequence that is at least about 35%,
preferably 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical
to the amino acid sequence of SEQ ID NO:33.
Also within the invention are isolated polypeptides or proteins
having an amino acid sequence that is at least about 35%,
preferably 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical
to the amino acid sequence of SEQ ID NO:41
Also within the invention are isolated polypeptides or proteins
having an amino acid sequence that is at least about 35%,
preferably 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical
to the amino acid sequence of SEQ ID NO:45.
Also within the invention are isolated polypeptides or proteins
which are encoded by a nucleic acid molecule having a nucleotide
sequence that is at least about 48%, preferably 50%, 55%, 60%, 65%,
75%, 85%, or 95% identical to the nucleic acid sequence encoding
SEQ ID NO:3, and isolated polypeptides or proteins which are
encoded by a nucleic acid molecule having a nucleotide sequence
which hybridizes under stringent hybridization conditions to a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1
or 2, a complement thereof, or the non-coding strand of the cDNA of
ATCC Accession Number 207116.
Also within the invention are isolated polypeptides or proteins
which are encoded by a nucleic acid molecule having a nucleotide
sequence that is at least about 30%, preferably 35%, 40%, 45%, 50%,
55%, 60%, 65%, 75%, 85%, or 95% identical to the nucleic acid
sequence encoding SEQ ID NO:15, and isolated polypeptides or
proteins which are encoded by a nucleic acid molecule having a
nucleotide sequence which hybridizes under stringent hybridization
conditions to a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:13 or 14, a complement thereof, or the
non-coding strand of the cDNA of ATCC Accession Number 207116.
Also within the invention are isolated polypeptides or proteins
which are encoded by a nucleic acid molecule having a nucleotide
sequence that is at least about 93%, preferably 94%, 95%, 96%, 97%,
or 98% identical to the nucleic acid sequence encoding SEQ ID
NO:21, and isolated polypeptides or proteins which are encoded by a
nucleic acid molecule having a nucleotide sequence which hybridizes
under stringent hybridization conditions to a nucleic acid molecule
having the nucleotide sequence of SEQ ID NO:19, 20, or 44, a
complement thereof, or the non-coding strand of the cDNA of ATCC
Accession Number 207116.
Also within the invention are isolated polypeptides or proteins
which are encoded by a nucleic acid molecule having a nucleotide
sequence that is at least about 30%, preferably 35%, 40%, 45%, 50%,
55%, 60%, 65%, 75%, 85%, or 95% identical to the nucleic acid
sequence encoding SEQ ID NO:33, and isolated polypeptides or
proteins which are encoded by a nucleic acid molecule having a
nucleotide sequence which hybridizes under stringent hybridization
conditions to a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:31 or 32, a complement thereof.
Also within the invention are isolated polypeptides or proteins
which are encoded by a nucleic acid molecule having a nucleotide
sequence that is at least about 30%, preferably 35%, 40%, 45%, 50%,
55%, 60%, 65%, 75%, 85%, or 95% identical to the nucleic acid
sequence encoding SEQ ID NO:41, and isolated polypeptides or
proteins which are encoded by a nucleic acid molecule having a
nucleotide sequence which hybridizes under stringent hybridization
conditions to a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:39 or 40.
Also within the invention are isolated polypeptides or proteins
which are encoded by a nucleic acid molecule having a nucleotide
sequence that is at least about 30%, preferably 35%, 40%, 45%, 50%,
55%, 60%, 65%, 75%, 85%, or 95% identical to the nucleic acid
sequence encoding SEQ ID NO:45, and isolated polypeptides or
proteins which are encoded by a nucleic acid molecule having a
nucleotide sequence which hybridizes under stringent hybridization
conditions to a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:19 or 44.
Also within the invention are polypeptides which are naturally
occurring allelic variants of a polypeptide that includes the amino
acid sequence of SEQ ID NO:3, 15, 21, 33, 41, or 45, or the amino
acid sequence encoded by the cDNA of ATCC Accession Number 207116,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule having the sequence of SEQ ID
NO:1, 2, 3, 13, 14, 19, 20, 31, 32, 39, 40, or 44 or a complement
thereof under stringent conditions.
The invention also features nucleic acid molecules that hybridize
under stringent conditions to a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1 or 2, the cDNA of ATCC Accession
Number 207116, or a complement thereof. In other embodiments, the
nucleic acid molecules are at least 310 (400, 500, 600, 800, 1000,
1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750,
4000, or 4020) nucleotides in length and hybridize under stringent
conditions to a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:1 or 2, the cDNA of ATCC Accession Number
207116, or a complement thereof.
The invention also features nucleic acid molecules that hybridize
under stringent conditions to a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:13 or 14, the cDNA of ATCC
Accession Number 207116, or a complement thereof. In other
embodiments, the nucleic acid molecules are at least 515 (530, 550,
600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2100, or 2150) nucleotides in length and
hybridize under stringent conditions to a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:13 or 14, the cDNA
of ATCC Accession Number 207116, or a complement thereof.
The invention also features nucleic acid molecules that hybridize
under stringent conditions to a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:19 or 20, or the cDNA of ATCC
Accession Number 207116, or a complement thereof. In other
embodiments, the nucleic acid molecules are at least 2220 (2260,
2300, 2340, 2380, 2420, 2460, 2500, 2540, 2580, 2620, 2660, 2700,
2740, 2780, or 2800) nucleotides in length and hybridize under
stringent conditions to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:19 or 20, the cDNA of ATCC
Accession Number 207116, or a complement thereof.
The invention also features nucleic acid molecules that hybridize
under stringent conditions to a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:31 or 32, or a complement thereof.
In other embodiments, the nucleic acid molecules are at least 100
(150, 200, 250, 300, 350, 400, 500, 600, or 700) nucleotides in
length and hybridize under stringent conditions to a nucleic acid
molecule comprising the nucleotide sequence of SEQ ID NO:31 or 32,
or a complement thereof.
The invention also features nucleic acid molecules that hybridize
under stringent conditions to a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:39 or 40, or a complement thereof.
In other embodiments, the nucleic acid molecules are at least 515
(530, 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,
1600, 1700, 1800, 1900, 2000, 2100, or 2150) nucleotides in length
and hybridize under stringent conditions to a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:39 or 40, or a
complement thereof.
The invention also features nucleic acid molecules that hybridize
under stringent conditions to a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:19 or 44, the cDNA of ATCC
Accession Number 207116, or a complement thereof. In other
embodiments, the nucleic acid molecules are at least 515 (530, 550,
600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2100, or 2150) nucleotides in length and
hybridize under stringent conditions to a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:19 or 44, the cDNA
of ATCC Accession Number 207116, or a complement thereof.
In one embodiment, the invention provides an isolated nucleic acid
molecule which is antisense to the coding strand of a nucleic acid
of the invention.
Another aspect of the invention provides vectors, e.g., recombinant
expression vectors, comprising a nucleic acid molecule of the
invention. In another embodiment, the invention provides host cells
containing such a vector or a nucleic acid molecule of the
invention. The invention also provides methods for producing a
polypeptide of the invention by culturing, in a suitable medium, a
host cell of the invention containing a recombinant expression
vector such that a polypeptide is produced.
Another aspect of this invention features isolated or recombinant
proteins and polypeptides of the invention. Preferred proteins and
polypeptides possess at least one biological activity possessed by
the corresponding naturally-occurring human polypeptide. An
activity, a biological activity, or a functional activity of a
polypeptide or nucleic acid of the invention refers to an activity
exerted by a protein, polypeptide or nucleic acid molecule of the
invention on a responsive cell as determined in vivo, or in vitro,
according to standard techniques. Such activities can be a direct
activity, such as an association with or an enzymatic activity on a
second protein, or an indirect activity, such as a cellular
signaling activity mediated by interaction of the protein with a
second protein.
For TANGO 228, biological activities include, e.g., (1) the ability
to modulate, e.g., stabilize, protein-protein interactions (e.g.,
homophilic and/or heterophilic), and protein-ligand interactions,
e.g., in receptor-ligand recognition; (2) ability to interact with
(e.g., noncovalently bind to) antigens, e.g., the antigens that
elicited their formation; (3) the ability to initiate the immune
response; and (4) the ability to modulate the activity (e.g., the
development and/or activation) of connective tissue cells (e.g.,
mast cells and/or monocytes).
Other activities of TANGO 228 include: (1) the ability to modulate
intracellular signaling cascades (e.g., signal transduction
cascade), e.g., by interacting with target peptides (e.g.,
phosphotyrosine-containing target peptides); and (2) the ability to
mediate (e.g., initiate) the allergic response, e.g., by serving as
a receptor to antigens and/or a signaling molecule to other immune
response mediators (e.g., histamines).
Still other activities of TANGO 228 include: (1) the ability to
modulate (e.g., inhibit) lipid associated processes (e.g.,
exocytosis and/or lipid mediator generation) by, e.g., interacting
with (e.g., binding to) a cell surface protein (e.g., a receptor)
on a cell type involved in the immune response (e.g., mast cell);
and (2) the ability to perform one or more of the functions of rat
surface protein MCA-32 described, for example, in Pirozzi et al.
(1995) Journal of Immunology. 155:5811 5818, the contents of which
are incorporated herein by reference.
For TANGO 240, biological activities include, e.g., (1) the ability
to modulate the tuberculosis pathology pathway in the same fashion
as Mycobacterium tuberculosis conserved hypothetical protein
Rv0712; and (2) the ability to modulate the function, migration,
proliferation (e.g., suppress cell growth), and/or differentiation
of cells, e.g., cells in tissues in which it is expressed (see
description of expression data below).
For TANGO 243, biological activities include, e.g., (1) the ability
to modulate (e.g., activate) the activity of enzymes (e.g.,
phospholipases) that hydrolyze lipids (e.g., phospholipids); (2)
the ability to modulate (e.g., activate) the activity of enzymes
that release precursors (e.g., arachidonic acid) of regulatory
molecules (e.g., prostaglandins and/or eicosanoids) associated with
the arthropathy pathway; (3) the ability to mediate (e.g.,
activate) the arthropathy pathway, e.g., through biological
activity (1); and (4) the ability to modulate (e.g., induce) the
activity (e.g., proliferation) of cell types associated with the
arthropathic pathway (e.g., leukocytes, e.g., polymorphonuclear
leukocytes, and/or mononuclear inflammatory cells).
Other activities of TANGO 243 include: (1) the ability to modulate,
e.g., by causing cells to alter (e.g., increase) the synthesis of,
and/or by causing cells (e.g., macrophages) to release, cell
mediating molecules, e.g., cytokines (e.g., IL-1 and/or TNF); (2)
the ability to modulate (e.g., initiate) an immune and/or
inflammatory response, e.g., through biological activity (1); and
(3) the ability to modulate (e.g., perpetuate) an immune and/or
inflammatory response, e.g., through biological activity (1),
whereby TANGO 243 itself is stimulated by the cell mediating
molecule that it modulates (e.g., cytokine (e.g., IL-1 and/or
TNF)).
Additional activities of TANGO 243 include: (1) the ability to
modulate (e.g., initiate) the activity of enzymes (e.g.,
phospholipases (e.g., phospholipase A2), e.g., by modulating their
signal transduction; (2) the ability to modulate (e.g., promote)
cell-cell interaction (e.g., chemotaxis) by modulating (e.g.,
initiating) phospolipase A2 activation and/or signal transduction;
(3) the ability to modulate (e.g., increase) cell permeability
(e.g., endothelial cell permeability), e.g., via biological
activity (2); and (4) the ability to modulate (e.g., promote)
cell-cell adhesion (e.g., membrane fusion), e.g., via biological
activity (2).
Still other activities of TANGO 243 include: (1) the ability to
modulate (e.g., inhibit) cellular functions, e.g., cell division,
cell-fate determination, gene transcription, transmembrane
signaling, mRNA modification, and/or vesicle fusion; (2) the
ability to modulate signal transduction, e.g., that of
transmembrane receptors; and (3) the ability to perform one or more
of the functions of human PLAP described, for example, in Bomalaski
et al. (1990) J Lab Clin Med. 16 (6):814 825, the contents of which
are incorporated herein by reference.
In one embodiment, a polypeptide of the invention has an amino acid
sequence sufficiently identical to an identified domain of a
polypeptide of the invention. As used herein, the term
"sufficiently identical" refers to a first amino acid or nucleotide
sequence which contains a sufficient or minimum number of identical
or equivalent (e.g., with a similar side chain) amino acid residues
or nucleotides to a second amino acid or nucleotide sequence such
that the first and second amino acid or nucleotide sequences have a
common structural domain and/or common functional activity. For
example, amino acid or nucleotide sequences which contain a common
structural domain having about 60% identity, preferably 65%
identity, more preferably 75%, 85%, 95%, 98% or more identity are
defined herein as sufficiently identical.
In one embodiment, a TANGO 228 protein includes at least one or
more of the following domains: a signal sequence, an extracellular
domain, an Ig domain, a transmembrane domain, and an intracellular
domain. In another embodiment, a TANGO 228 protein includes an
extracellular domain, two Ig domains, and is wholly secreted. In
yet another, a TANGO 228 protein includes an extracellular domain,
two Ig domains, a transmembrane domain, and a cytoplasmic domain,
and is a transmembrane protein.
In one embodiment, a TANGO 240 protein includes a signal
peptide.
In one embodiment, a TANGO 243 protein includes a G-beta domain. In
another embodiment, a TANGO 243 protein includes at least about two
G-beta domains. In still another embodiment, a TANGO 243 protein
includes at least three, four, five, six, or seven G-beta
domains.
The polypeptides of the present invention, or biologically active
portions thereof, can be operably linked to a heterologous amino
acid sequence to form fusion proteins. The invention further
features antibodies that specifically bind a polypeptide of the
invention such as monoclonal or polyclonal antibodies. In addition,
the polypeptides of the invention or biologically active portions
thereof can be incorporated into pharmaceutical compositions, which
optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides methods for
detecting the presence of the activity or expression of a
polypeptide of the invention in a biological sample by contacting
the biological sample with an agent capable of detecting an
indicator of activity such that the presence of activity is
detected in the biological sample.
In another aspect, the invention provides methods for modulating
activity of a polypeptide of the invention comprising contacting a
cell with an agent that modulates (inhibits or stimulates) the
activity or expression of a polypeptide of the invention such that
activity or expression in the cell is modulated. In one embodiment,
the agent is an antibody that specifically binds to a polypeptide
of the invention.
In another embodiment, the agent modulates expression of a
polypeptide of the invention by modulating transcription, splicing,
or translation of an mRNA encoding a polypeptide of the invention.
In yet another embodiment, the agent is a nucleic acid molecule
having a nucleotide sequence that is antisense to the coding strand
of an mRNA encoding a polypeptide of the invention.
The present invention also provides methods to treat a subject
having a disorder characterized by aberrant activity of a
polypeptide of the invention or aberrant expression of a nucleic
acid of the invention by administering an agent which is a
modulator of the activity of a polypeptide of the invention or a
modulator of the expression of a nucleic acid of the invention to
the subject. In one embodiment, the modulator is a protein of the
invention. In another embodiment, the modulator is a nucleic acid
of the invention. In other embodiments, the modulator is a peptide,
peptidomimetic, or other small molecule.
The present invention also provides diagnostic assays for
identifying the presence or absence of a genetic lesion or mutation
characterized by at least one of: (i) aberrant modification or
mutation of a gene encoding a polypeptide of the invention, (ii)
mis-regulation of a gene encoding a polypeptide of the invention,
and (iii) aberrant post-translational modification of the invention
wherein a wild-type form of the gene encodes a protein having the
activity of the polypeptide of the invention.
In another aspect, the invention provides a method for identifying
a compound that binds to or modulates the activity of a polypeptide
of the invention. In general, such methods entail measuring a
biological activity of the polypeptide in the presence and absence
of a test compound and identifying those compounds which alter the
activity of the polypeptide.
The invention also features methods for identifying a compound
which modulates the expression of a polypeptide or nucleic acid of
the invention by measuring the expression of the polypeptide or
nucleic acid in the presence and absence of the compound.
Other features and advantages of the invention will be apparent
from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A 1C depict the cDNA sequence of human TANGO 228 (SEQ ID
NO:1) and the predicted amino acid sequence of TANGO 228 (SEQ ID
NO:3). The open reading frame of SEQ ID NO:1 extends from
nucleotide 34 to nucleotide 1062 of SEQ ID NO:1 (SEQ ID NO:2).
FIG. 2 depicts a hydropathy plot of human TANGO 228. Relatively
hydrophobic regions of the protein are above the dashed horizontal
line, and relatively hydrophilic regions of the protein are below
the dashed horizontal line. The cysteine residues (cys) and
potential N-glycosylation sites (Ngly) are indicated by short
vertical lines just below the hydropathy trace. The dashed vertical
line separates the signal sequence (amino acids 1 to 19 of SEQ ID
NO:3; SEQ ID NO:5) on the left from the mature protein (amino acids
20 to 343 of SEQ ID NO:3; SEQ ID NO:4) on the right. Thicker gray
horizontal bars below the dashed horizontal line indicate
extracellular ("out"), transmembrane ("TM"), and intracellular
("in") regions of the molecule. Below the hydropathy plot, the
amino acid sequence of TANGO 228 is depicted.
FIGS. 3A 3H depict an alignment of the nucleotide sequence of rat
surface protein MCA-32 (SEQ ID NO:11; GenBank Accession Number
U39546) and the nucleotide sequence of human TANGO 228 (SEQ ID
NO:1). The nucleotide sequences of rat MCA-32 and human TANGO 228
are 28.3% identical. This alignment was performed using the ALIGN
alignment program with a PAM120 scoring matrix, a gap length
penalty of 12, and a gap penalty of 4.
FIGS. 4A 4C depict an alignment of the nucleotide sequence of the
open reading frames of rat MCA-32 (nucleotides 8 to 826 of SEQ ID
NO:11) and human TANGO 228 (SEQ ID NO:2). The nucleotide sequences
of the open reading frames of rat MCA-32 and human TANGO 228 (SEQ
ID NO:2) are 45.4% identical. This alignment was performed using
the ALIGN alignment program with a PAM120 scoring matrix, a gap
length penalty of 12, and a gap penalty of 4.
FIG. 5 depicts an alignment of the amino acid sequence of rat
MCA-32 (SEQ ID NO:12) and the amino acid sequence of human TANGO
228 (SEQ ID NO:3). The amino acid sequences of rat MCA-32 and human
TANGO 228 are 26.8% identical. This alignment was performed using
the ALIGN alignment program with a PAM120 scoring matrix, a gap
length penalty of 12, and a gap penalty of 4.
FIGS. 6A 6B depict the cDNA sequence of human TANGO 240 (SEQ ID
NO:13) and the predicted amino acid sequence of TANGO 240 (SEQ ID
NO:15). The open reading frame of SEQ ID NO:13 extends from
nucleotide 2 to nucleotide 1123 of SEQ ID NO:13 (SEQ ID NO:14).
FIG. 7 depicts a hydropathy plot of human TANGO 240. Relatively
hydrophobic regions of the protein are shown above the horizontal
line, and relatively hydrophilic regions of the protein are below
the horizontal line. The cysteine residues (cys) and potential
N-glycosylation sites (Ngly) are indicated by short vertical lines
just below the hydropathy trace. The dashed vertical line separates
the signal sequence (amino acids 1 to 34 of SEQ ID NO:15; SEQ ID
NO:17) on the left from the mature protein (amino acids 35 to 374
of SEQ ID NO:15; SEQ ID NO:16) on the right. Below the hydropathy
plot, the amino acid sequence of TANGO 240 is depicted.
FIG. 8 depicts an alignment of the amino acid sequence of the
Mycobacterium tuberculosis conserved hypothetical protein Rv0712
(SEQ ID NO:18; GenBank Accession Number Z84395) and the amino acid
sequence of human TANGO 240 (SEQ ID NO:15). The amino acid
sequences of Rv0712 and human TANGO 240 are 31.2% identical. This
alignment was performed using the ALIGN alignment program with a
PAM120 scoring matrix, a gap length penalty of 12, and a gap
penalty of 4.
FIGS. 9A 9C depict the cDNA sequence of human TANGO 243 (SEQ ID
NO:19) and the predicted amino acid sequence of TANGO 243 (SEQ ID
NO:21). The open reading frame of SEQ ID NO:19 extends from
nucleotide 183 to nucleotide 2567 of SEQ ID NO:19 (SEQ ID
NO:20).
FIG. 10 depicts a hydropathy plot of human TANGO 243. Relatively
hydrophobic regions of the protein are above the dashed horizontal
line, and relatively hydrophilic regions of the protein are below
the dashed horizontal line. The cysteine residues (cys) and
potential N-glycosylation sites (Ngly) are indicated by short
vertical lines just below the hydropathy trace. Below the
hydropathy plot, the amino acid sequence of TANGO 243 is
depicted.
FIGS. 11A 11F depict an alignment of the nucleotide sequence of
human PLAP (SEQ ID NO:29; GenBank Accession Number AF083395) and
the nucleotide sequence of human TANGO 243 (SEQ ID NO:19). The
nucleotide sequences of human PLAP and human TANGO 243 are 78.8%
identical. This alignment was performed using the ALIGN alignment
program with a PAM120 scoring matrix, a gap length penalty of 12,
and a gap penalty of 4.
FIGS. 12A 12E depict an alignment of the nucleotide sequence of the
open reading frames of human PLAP (nucleotides 1 to 2217 of SEQ ID
NO:29) and human TANGO 243 (SEQ ID NO:20). The nucleotide sequences
of the open reading frames of human PLAP and human TANGO 243 (SEQ
ID NO:20) are 92.7% identical. This alignment was performed using
the ALIGN alignment program with a PAM120 scoring matrix, a gap
length penalty of 12, and a gap penalty of 4.
FIGS. 13A 13B depict an alignment of the amino acid sequence of
human PLAP (SEQ ID NO:30) and the amino acid sequence of human
TANGO 243 (SEQ ID NO:21). The amino acid sequences of human PLAP
and human TANGO 243 are 92.8% identical. This alignment was
performed using the ALIGN alignment program with a PAM120 scoring
matrix, a gap length penalty of 12, and a gap penalty of 4.
FIG. 14 depicts the cDNA sequence of murine TANGO 228 (SEQ ID
NO:31) and the predicted amino acid sequence of murine TANGO 228
(SEQ ID NO:33). The open reading frame of SEQ ID NO:31 extends from
nucleotide 27 to nucleotide 743 of SEQ ID NO:31 (SEQ ID NO:33).
FIGS. 15A 15B depict an alignment of the nucleotide sequence of
murine TANGO 228 (SEQ ID NO:31; upper line of each pair) and the
nucleotide sequence of human TANGO 228 (SEQ ID NO:1; lower line of
each pair). The nucleotide sequences of murine TANGO 228 and human
TANGO 228 are 58.2% identical, over the length of the murien cDNA.
This alignment was performed using the ALIGN alignment program with
a PAM120 scoring matrix, a gap length penalty of 12, and a gap
penalty of 4.
FIGS. 16A 16B depict an alignment of the nucleotide sequence of the
open reading frames of murine TANGO 228 (SEQ ID NO:33; upper line
of each pair) and human TANGO 228 (SEQ ID NO:3; lower line of each
pair). The nucleotide sequences of the open reading frames of
murine TANGO 228 human TANGO 228 are 58.2% identical, over the
length of the murine ORF. This alignment was performed using the
ALIGN alignment program with a PAM120 scoring matrix, a gap length
penalty of 12, and a gap penalty of 4.
FIG. 17 depicts an alignment of the amino acid sequence of murine
TANGO 228 (SEQ ID NO:32; upper line of each pair) and human TANGO
228 (SEQ ID NO:3; lower line of each pair). The amino acid
sequences of murine TANGO 228 and human TANGO 228 are 30.6%
identical, over the length of the murine protein. This alignment
was performed using the ALIGN alignment program with a PAM120
scoring matrix, a gap length penalty of 12, and a gap penalty of
4.
FIGS. 18A 18B depict the cDNA sequence of murine TANGO 240 (SEQ ID
NO:39) and the predicted amino acid sequence of murine TANGO 240
(SEQ ID NO:41). The open reading frame of SEQ ID NO:41 extends from
nucleotide 2 to nucleotide 1117 of SEQ ID NO:41 (SEQ ID NO:40).
FIGS. 19A 19D depict an alignment of the nucleotide sequence of
murine TANGO 240 (SEQ ID NO:39; upper line of each pair) and the
nucleotide sequence of human TANGO 228 (SEQ ID NO:13; lower line of
each pair). In this alignment the sequences are 78.4% identical,
over the length of the human cDNA. This alignment was performed
using the ALIGN alignment program with a PAM120 scoring matrix, a
gap length penalty of 12, and a gap penalty of 4.
FIGS. 20A 20B depict an alignment of the nucleotide sequence of the
open reading frames of murine TANGO 240 (SEQ ID NO:40; upper line
of each pair) and human TANGO 240 (SEQ ID NO:14; lower line of each
pair). The nucleotide sequences of the open reading frames of
murine TANGO 240 human TANGO 240 are 84.4% identical. This
alignment was performed using the ALIGN alignment program with a
PAM120 scoring matrix, a gap length penalty of 12, and a gap
penalty of 4.
FIG. 21 depicts an alignment of the amino acid sequence of murine
TANGO 240 (SEQ ID NO:41; upper line of each pair) and human TANGO
240 (SEQ ID NO:17; lower line of each pair). The amino acid
sequences of murine TANGO 240 and human TANGO 240 are 86%
identical. This alignment was performed using the ALIGN alignment
program with a PAM120 scoring matrix, a gap length penalty of 12,
and a gap penalty of 4.
FIGS. 22A 22C depict the cDNA sequence of human TANGO 243 (SEQ ID
NO:19) and the predicted amino acid sequence of a longer form of
TANGO 243 (SEQ ID NO:45) arising from an alternative translation of
the human TANGO 243 cDNA. In this translation the open reading
frame of SEQ ID NO:19 extends from nucleotide 3 to nucleotide 2567
of SEQ ID NO:19 (SEQ ID NO:44).
FIG. 23 depicts a hydropathy plot of the longer form of human TANGO
243 arising from the alternative translation of the human TANGO 243
cDNA. Relatively hydrophobic regions of the protein are above the
dashed horizontal line, and relatively hydrophilic regions of the
protein are below the dashed horizontal line. The cysteine residues
(cys) and potential N-glycosylation sites (Ngly) are indicated by
short vertical lines just below the hydropathy trace. Below the
hydropathy plot, the amino acid sequence of TANGO 243 is
depicted.
DETAILED DESCRIPTION OF THE INVENTION
The TANGO 228, TANGO 240, and TANGO 243 proteins and nucleic acid
molecules comprise a family of molecules having certain conserved
structural and functional features. As used herein, the term
"family" is intended to mean two or more proteins or nucleic acid
molecules having a common structural domain and having sufficient
amino acid or nucleotide sequence identity as defined herein.
Family members can be from either the same or different species.
For example, a family can comprises two or more proteins of human
origin, or can comprise one or more proteins of human origin and
one or more of non-human origin. Members of the same family may
also have common structural domains.
For example, TANGO 228 proteins and TANGO 240 proteins of the
invention have signal sequences. As used herein, a "signal
sequence" includes a peptide of at least about 15 or 20 amino acid
residues in length which occurs at the N-terminus of secretory and
membrane-bound proteins and which contains at least about 70%
hydrophobic amino acid residues such as alanine, leucine,
isoleucine, phenylalanine, proline, tyrosine, tryptophan, or
valine. In a preferred embodiment, a signal sequence contains at
least about 10 to 40 amino acid residues, preferably about 19 34
amino acid residues, and has at least about 60 80%, more preferably
65 75%, and more preferably at least about 70% hydrophobic
residues. A signal sequence serves to direct a protein containing
such a sequence to a lipid bilayer. Thus, in one embodiment, a
TANGO 228 protein contains a signal sequence at about amino acids 1
to 19 of SEQ ID NO:3 (SEQ ID NO:5). In another embodiment, a TANGO
240 protein contains a signal sequence at about amino acids 1 to 34
of SEQ ID NO:15 (SEQ ID NO:17). The signal sequence is cleaved
during processing of the mature protein.
In another example, a TANGO 228 family member also includes one or
more of the following domains: (1) an extracellular domain; (2) a
transmembrane domain; and (3) a cytoplasmic domain. Thus, in one
embodiment, a TANGO 228 protein contains an extracellular domain at
about amino acids 1 to 227 of SEQ ID NO:3 (SEQ ID NO:6). In another
embodiment, a TANGO 228 protein contains a transmembrane domain at
about amino acids 228 to 249 of SEQ ID NO:3 (SEQ ID NO:7). In
another embodiment, a TANGO 228 protein contains a cytoplasmic
domain at about amino acids 250 to 343 of SEQ ID NO:3 (SEQ ID
NO:10).
In one embodiment, the extracellular domain of TANGO 228 can also
include an Ig domain. In another embodiment, the extracellular
domain of TANGO 228 includes about 1 to 10, preferably about 3 8,
more preferably about 6, N-glycosylation sites, about 1 to 30,
preferably about 10 to 20, more preferably about 15 conserved
serine residues (not including residues within the Ig domain), and
about 1 to 20, preferably about 5 to 15, more preferably about 11
conserved threonine residues (not including residues within the Ig
domain).
In a preferred embodiment, a TANGO 228 family member has the amino
acid sequence of SEQ ID NO:3 wherein the extracellular domain is
located at about amino acids 1 to 227 (SEQ ID NO:6), the
N-glycosylation sites are located at about amino acid residue
positions 51 to 54, 60 to 63, 89 to 92, 151 to 154, 157 to 160, and
182 to 185, the conserved serine residues are located at about
amino acid positions 3, 6, 12, 15, 16, 36, 41, 109, 111, 114, 118,
127, 209, 216, and 221, and the conserved threonine residues are
located at about amino acid positions 18, 31, 43, 108, 120, 126,
137, 139, 207, 213, and 217.
TANGO 228 family members can also include an Ig domain contained
within the extracellular domain. As used herein, the term "Ig
domain" refers to a protein domain bearing homology to
immunoglobulin superfamily members. An Ig domain includes about 30
90 amino acid residues, preferably about 40 80 amino acid residues,
more preferably about 50 70 amino acid residues, still more
preferably about 55 65 amino acid residues, and most preferably
about 57 to 59 amino acid residues. Typically, an Ig domain
contains a conserved cysteine residue within about 5 to 15 amino
acid residues, preferably about 7 to 12 amino acid residues, and
most preferably about 8 amino acid residues from its N-terminal
end, and another conserved cysteine residue within about 1 to 5
amino acid residues, preferably about 2 to 4 amino acid residues,
and most preferably about 3 amino acid residues from its C-terminal
end.
An Ig domain typically has the following consensus sequence,
beginning about 1 to 15 amino acid residues, more preferably about
3 to 10 amino acid residues, and most preferably about 5 amino acid
residues from the domain C terminal end of a protein:
(FY)-Xaa-C-Xaa-(VA)-COO-, wherein (FY) is either a phenylalanine or
a tyrosine residue (preferably tyrosine), Xaa is any amino acid, C
is a cysteine residue, (VA) is either a valine or an alanine
residue (preferably alanine), and COO-- is the protein C
terminus.
In one embodiment, a TANGO 228 family member includes one or more
Ig domains having an amino acid sequence that is at least about
55%, preferably at least about 65%, more preferably at least about
75%, yet more preferably at least about 85%, and most preferably at
least about 95% identical to amino acids 49 to 105 and/or amino
acids 140 to 198 of SEQ ID NO:3, which are the Ig domains of TANGO
228 (these Ig domains are also represented as SEQ ID NO:8 and 9,
respectively). In another embodiment, a TANGO 228 family member
includes one or more Ig domains having an amino acid sequence that
is at least about 55%, preferably at least about 65%, more
preferably at least about 75%, yet more preferably at least about
85%, and most preferably at least about 95% identical to amino
acids 49 to 105 and/or amino acids 140 to 198 of SEQ ID NO:3 (SEQ
ID NO:8 and 9, respectively), includes a conserved cysteine residue
about 8 residues downstream from the N-terminus of the Ig domain,
and has one or more Ig domain consensus sequences described herein.
In another embodiment, a TANGO 228 family member includes one or
more Ig domains having an amino acid sequence that is at least 55%,
preferably at least about 65%, more preferably at least about 75%,
yet more preferably at least about 85%, and most preferably at
least about 95% identical to amino acids 49 to 105 and/or amino
acids 140 to 198 of SEQ ID NO:3 (SEQ ID NO:8 and 9, respectively),
includes a conserved cysteine residue 8 residues downstream from
the N-terminus of the Ig domain, has one or more Ig domain
consensus sequences described herein, and has a conserved cysteine
within the consensus sequence that forms a disulfide both with said
first conserved cysteine. In yet another embodiment, a TANGO 228
family member includes one or more Ig domains having an amino acid
sequence that is at least 55%, preferably at least about 65%, more
preferably at least about 75%, yet more preferably at least about
85%, and most preferably at least about 95% identical to amino
acids 49 to 105 and/or amino acids 140 to 198 of SEQ ID NO:3 (SEQ
ID NO:8 and 9, respectively), includes a conserved cysteine residue
8 residues downstream from the N-terminus of the Ig domain, has one
or more Ig domain consensus sequences described herein, has a
conserved cysteine within the consensus sequence that forms a
disulfide both with said first conserved cysteine, and has at least
one TANGO 228 biological activity as described herein.
In a preferred embodiment, a TANGO 228 family member has the amino
acid sequence of SEQ ID NO:3 wherein the aforementioned Ig domain
conserved residues are located as follows: the N-terminal conserved
cysteine residue is located at about amino acid residue position 56
(within the first Ig domain (SEQ ID NO:8)) and at about amino acid
residue position 147 (within the second Ig domain (SEQ ID NO:9)),
and the C-terminal conserved cysteine residue is located at about
amino acid position 103 (within the first Ig domain (SEQ ID NO:8))
and at about amino acid residue position 196 (within the second Ig
domain (SEQ ID NO:9)).
A TANGO 228 family member can also include a cytoplasmic domain.
The cytoplasmic domain can include hydrophobic amino acid residues,
and can contain several SH2 domain recognition sites, which contain
typically begin with a tyrosine residue. This cytoplasmic domain
can contain about 1 to 5, more preferably about 2 to 4, and still
more preferably about 3 SH2 domain recognition sites.
One type of SH2 domain recognition site typically has the following
consensus sequence: Y-Xaa1-Xaa1-(IP), wherein Y is a tyrosine
residue, Xaa1 is any hydrophobic amino acid, and (IP) is either an
isoleucine or proline residue. Another type of SH2 domain
recognition site typically has the following consensus sequence:
Y-Xaa1-Xaa-Xaa1, wherein Y is a tyrosine residue, Xaa1 is any
hydrophobic amino acid, and Xaa is any amino acid.
In a preferred embodiment, a TANGO 228 family member has the amino
acid sequence of SEQ ID NO:3 wherein the cytoplasmic domain is
located at about amino acids 250 to 343 (SEQ ID NO:10) and the
conserved SH2 domain recognition sites are located at about amino
acid positions 276 to 279, 313 to 317, and 338 to 341.
A TANGO 240 family member can include a signal sequence. In a
preferred embodiment, a TANGO 240 family member has the amino acid
sequence of SEQ ID NO:15, and the signal sequence is located at
about amino acids 1 to 34.
A TANGO 243 family member can include one or more G-beta domains.
As used herein, a "G-beta domain" refers to a domain which includes
about 25 to 55 amino acid residues, preferably about 30 to 50 amino
acid residues, more preferably about 35 55 amino acid residues, and
most preferably about 38 to 44 amino acid residues. In addition, a
G-beta domain contains a conserved glycine residue adjacent to a
conserved histidine residue, a conserved aspartic acid residue, and
one or more conserved valine or isoleucine residues.
G-beta domains are typically found within the WD-repeat
(tryptophan-aspartate repeat) family of proteins. The WD-repeat
family of proteins are found in all eukaryotes but not in
prokaryotes, and are functionally diverse, in some cases regulating
cellular functions, transmembrane signaling, and/or vesicle fusion,
among other things.
A G-beta domain typically has the following consensus sequence:
G-H-Xaa(n1)-V-Xaa(n2)-[V or L]-Xaa(n3)-D-Xaa(n4)-W, wherein G is
glycine, H is histidine, V is valine, D is aspartic acid, L is
leucine, W is tryptophan, Xaa is any amino acid, n1 is about 1 8
amino acid residues, more preferably about 2 5 amino acid residues,
and more preferably about 3 amino acid residues, n2 is about 1 8
amino acid residues, more preferably about 1 4 amino acid residues,
and more preferably about 2 amino acid residues, n3 is about 5 30
amino acid residues, more preferably about 10 25 amino acid
residues, and more preferably about 14 19 amino acid residues, and
n4 is about 1 8 amino acid residues, more preferably about 3 6
amino acid residues, and more preferably about 5 amino acid
residues.
A TANGO 243 family member can also include a G-beta-like domain. As
used herein, a "G-beta-like domain" refers to an stretch of amino
acid residues, about 25 to 55 residues, preferably about 30 to 50
residues, more preferably about 35 55 amino acid residues, and most
preferably about 38 to 44 residues, in length, that is similar in
content to a G-beta domain. A G-beta-like domain typically has the
following consensus sequence: [V or I]-Xaa(2)-[V or C]-Xaa(n1)-[P
or D]-[D or N]-G-Xaa(n2)-G-Xaa(2)-D, wherein V is valine, I is
isoleucine, C is cysteine, P is proline, D is aspartic acid, G is
glycine, Xaa is any amino acid, n1 is about 1 to 8 amino acid
residues, preferably about 2 to 5 amino acid residues, more
preferably about 3 amino acid residues, and n2 is about 1 to 8
amino acid residues, preferably about 2 to 5 amino acid residues,
more preferably about 4 amino acid residues.
In one embodiment, a TANGO 243 family member includes one or more
G-beta domains having an amino acid sequence that is at least about
55%, preferably at least about 65%, more preferably at least about
75%, yet more preferably at least about 85%, and most preferably at
least about 95% identical to amino acids 8 to 47, or amino acids 55
to 98, or amino acids 102 to 139, or amino acids 141 to 179, or
amino acids 181 to 218, or amino acids 221 to 259 of SEQ ID NO:21,
which are the G-beta domains of TANGO 243 (these G-beta domains are
also represented as SEQ ID NO:22, 23, 24, 25, 26, and 27,
respectively). In another embodiment, a TANGO 243 family member
includes one or more G-beta domains having an amino acid sequence
that is at least about 55%, preferably at least about 65%, more
preferably at least about 75%, yet more preferably at least about
85%, and most preferably at least about 95% identical to amino
acids 8 to 47, or amino acids 55 to 98, or amino acids 102 to 139,
or amino acids 141 to 179, or amino acids 181 to 218, or amino
acids 221 to 259 of SEQ ID NO:21 (SEQ ID NO:22, 23, 24, 25, 26, and
27, respectively), and has a G-beta domain consensus sequence as
described herein. In yet another embodiment, a TANGO 243 family
member includes one or more G-beta domains having an amino acid
sequence that is at least about 55%, preferably at least about 65%,
more preferably at least about 75%, yet more preferably at least
about 85%, and most preferably at least about 95% identical to
amino acids 8 to 47, or amino acids 55 to 98, or amino acids 102 to
139, or amino acids 141 to 179, or amino acids 181 to 218, or amino
acids 221 to 259 of SEQ ID NO:21 (SEQ ID NO:22, 23, 24, 25, 26, and
27, respectively), has a G-beta domain consensus sequence as
described herein, and has at least one TANGO 243 biological
activity as described herein.
In another embodiment, a TANGO 243 family member includes one or
more G-beta domains having an amino acid sequence that is at least
about 55%, preferably at least about 65%, more preferably at least
about 75%, yet more preferably at least about 85%, and most
preferably at least about 95% identical to amino acids 8 to 47, or
amino acids 55 to 98, or amino acids 102 to 139, or amino acids 141
to 179, or amino acids 181 to 218, or amino acids 221 to 259 of SEQ
ID NO:21, amino acids 68 to 107, or amino acids 115 to 158, or
amino acids 162 to 199, or amino acids 201 to 139, or amino acids
141 to 278, or amino acids 281 to 219 of SEQ ID NO:45 (SEQ ID
NO:22, 23, 24, 25, 26, and 27, respectively), G-beta-like domain
having an amino acid sequence that is at least about 55%,
preferably at least about 65%, more preferably at least about 75%,
yet more preferably at least about 85%, and most preferably at
least about 95% identical to amino acids 261 to 298 of SEQ ID NO:21
(SEQ ID NO:28), has a G-beta domain consensus sequence and a
G-beta-like domain consensus sequence as described herein, and has
at least one TANGO 243 biological activity as described herein.
A TANGO 243 family member can include a PLAP-like domain. A
PLAP-like domain 1 can include at least 20, 25, 30, or 40
contiguous amino acids of SEQ ID NO:47. A PLAP-like domain 2 can
include at least 20, 25, 30, 40, or 45 contiguous amino acids of
SEQ ID NO:48. A PLAP-like domain 3 can include 20, 25, 30, 40, 45,
50, 60, 70, or 80 contiguous amino acids of SEQ ID NO:49. A
PLAP-like domain 1 can include at least 20, 25, 30, or 35
contiguous amino acids of SEQ ID NO:50.
In a preferred embodiment, a TANGO 243 family member has the amino
acid sequence of SEQ ID NO:21 wherein the G-beta consensus
sequences are located from amino acid 16 to 46 (the first G-beta
domain (SEQ ID NO:22)), 63 to 97 (the second G-beta domain (SEQ ID
NO:23)), 110 to 138 (the third G-beta domain (SEQ ID NO:24)), 149
to 178 (the fourth G-beta domain (SEQ ID NO:25)), 189 to 217 (the
fifth G-beta domain (SEQ ID NO:26)), and 229 to 258 (the sixth
G-beta domain (SEQ ID NO:27)), and the G-beta-like consensus
sequence is located from amino acid 261 to 298 (SEQ ID NO:28).
Various features of human TANGO 228, murine TANGO 228, human TANGO
240, and human TANGO 243 are summarized below.
Human TANGO 228
A cDNA encoding human TANGO 228 was identified by analyzing the
sequences of clones present in a fetal spleen cDNA library. This
analysis led to the identification of a clone, jthsa055f08,
encoding full-length human TANGO 228. The human TANGO 228 cDNA of
this clone is 4043 nucleotides long (FIGS. 1A 1C; SEQ ID NO:1). The
open reading frame of this cDNA, nucleotides 34 to 1062 of SEQ ID
NO:1 (SEQ ID NO:2), encodes a 343 amino acid transmembrane protein
(FIGS. 1A 1C; SEQ ID NO:3).
The signal peptide prediction program SIGNALP (Nielsen et al.
(1997) Protein Engineering 10:1 6) predicted that human TANGO 228
includes a 19 amino acid signal peptide (amino acid 1 to about
amino acid 19 of SEQ ID NO:3)(SEQ ID NO:5) preceding the mature
TANGO 228 protein (corresponding to about amino acid 20 to amino
acid 343 of SEQ ID NO:3)(SEQ ID NO:4). The TANGO 228 protein
molecular weight is 38.7 kDa prior to the cleavage of the signal
peptide, 36.4 kDa after cleavage of the signal peptide.
TANGO 228 includes a extracellular domain (about amino acids 1 to
227 of SEQ ID NO:3; SEQ ID NO:6), a transmembrane domain (about
amino acids 228 to 249 of SEQ ID NO:3; SEQ ID NO:7), two Ig domains
(about amino acids 49 to 105, and about amino acids 140 to 198, of
SEQ ID NO:3; SEQ ID NO:8 and 9), and a cytoplasmic domain (about
amino acids 250 to 343 of SEQ ID NO:3; SEQ ID NO:10).
An N-glycosylation site having the sequence NVSM is found from
amino acids 51 to 54 of SEQ ID NO:3. A second N-glycosylation site
having the sequence NKSL is found from amino acids 60 to 63. A
third N-glycosylation site having the sequence NLSI is found from
amino acids 89 to 92. A fourth N-glycosylation site having the
sequence NGSL is found from amino acids 151 to 154. A fifth
N-glycosylation site having the sequence NYTF is found from amino
acids 157 to 160. A sixth N-glycosylation site having the sequence
NLTK is found from amino acids 182 to 185. A cAMP and
cGMP-dependent protein kinase phosphorylation site having the
sequence RRKT is found from amino acids 71 to 74. A protein kinase
C phosphorylation site having the sequence TCR is found from amino
acids 18 to 20. A second protein kinase C phosphorylation site
having the sequence SHK is found from amino acids 57 to 59. A third
protein kinase C phosphorylation site having the sequence TDR is
found from amino acids 139 to 141. A fourth protein kinase C
phosphorylation site having the sequence TKK is found from amino
acids 184 to 186. A fifth protein kinase C phosphorylation site
having the sequence TRK is found from amino acids 254 to 256. A
sixth protein kinase C phosphorylation site having the sequence SYK
is found from amino acids 331 to 333. A casein kinase II
phosphorylation site having the sequence SITE is found from amino
acids 91 to 94. A second casein kinase II phosphorylation site
having the sequence TIVD is found from amino acids 120 to 123. A
third casein kinase II phosphorylation site having the sequence
TETD is found from amino acids 137 to 140. A fourth casein kinase
II phosphorylation site having the sequence TFFE is found from
amino acids 159 to 162. A fifth casein kinase II phosphorylation
site having the sequence SKYD is found from amino acids 172 to 175.
A sixth casein kinase II phosphorylation site having the sequence
TGGD is found from amino acids 217 to 220. A seventh casein kinase
II phosphorylation site having the sequence TAME is found from
amino acids 269 to 272. An eighth casein kinase II phosphorylation
site having the sequence SVPE is found from amino acids 288 to 291.
A ninth casein kinase II phosphorylation site having the sequence
TAQD is found from amino acids 300 to 303. A tyrosine kinase
phosphorylation site having the sequence KNPGEEEEY is found from
amino acids 186 to 194. A second tyrosine kinase phosphorylation
site having the sequence KHSQELQY is found from amino acids 306 to
313. An N-myristoylation site having the sequence GQNVSM is found
from amino acids 49 to 54. A second N-myristoylation site having
the sequence GTQDGK is found from amino acids 77 to 82. A third
N-myristoylation site having the sequence GIYANI is found from
amino acids 274 to 279. A fourth N-myristoylation site having the
sequence GSRPCV is found from amino acids 293 to 298. A cell
attachment sequence having the sequence RGD is found from amino
acids 266 to 268. A leucine zipper pattern having the sequence
LLLPGLLLLLVVIILILAFWVL is found from amino acids 228 to 249.
TANGO 228 is homologous to rat MCA-32, which is known to exist in
two forms, one of which has a transmembrane domain, and the other
which does not. Thus TANGO 228, like rat MCA-32, is likely to exist
in two forms, one having domains as follows and is wholly secreted:
an extracellular domain (SEQ ID NO:6) containing one or more Ig
domains (SEQ ID NO:8 and 9), and the other having the following
domains and is a transmembrane, e.g., a cell surface, protein: an
extracellular domain (SEQ ID NO:6) containing one or more Ig
domains (SEQ ID NO:8 and 9), a transmembrane domain (SEQ ID NO:7),
and cytoplasmic domain (SEQ ID NO:10).
FIGS. 3A 3H show an alignment of the human TANGO 228 full length
nucleic acid sequence (SEQ ID NO:1) with the rat MCA-32 full length
nucleic acid sequence (SEQ ID NO:11). FIGS. 4A 4C show an alignment
of the human TANGO 228 coding region (SEQ ID NO:2) with the rat
MCA-32 coding region. FIG. 5 shows an alignment of the human TANGO
228 protein sequence (SEQ ID NO:3) with the rat MCA-32 protein
sequence (SEQ ID NO:12). As shown in FIG. 5, the human TANGO 228
signal sequence is represented by amino acids 1 19 (and encoded by
nucleotides 34 to 90 of SEQ ID NO:1). Though the rat MCA-32 gene
structure suggests it can function as a secreted protein or cell
surface protein, depending on the splice variant, it does not
exhibit the characteristic hydrophobic signal sequence generally
found in secretory and transmembrane proteins. The human TANGO 228
extracellular domain sequence (SEQ ID NO:6) is represented by amino
acids 1 to 227 (and encoded by nucleotides 34 to 714 of SEQ ID
NO:1), and the rat MCA-32 extracellular domain sequence is
represented by amino acids 1 to 177 (and encoded by nucleotides 8
to 523 of SEQ ID NO:11). The human TANGO 228 Ig domains (SEQ ID
NO:8 and 9) are represented by amino acids 49 to 105, and 140 to
198 (and encoded by nucleotides 178 to 348, and 451 to 627 of SEQ
ID NO:1), and the rat MCA-32 Ig domain is represented by amino
acids 99 to 153 (and encoded by nucleotides 287 to 451 of SEQ ID
NO:11). The human TANGO 228 transmembrane domain (SEQ ID NO:7) is
represented by amino acids 228 to 249 (and encoded by nucleotides
715 to 780 of SEQ ID NO:1), and the rat MCA-32 transmembrane domain
is represented by amino acids 178 to 199 (and encoded by
nucleotides 524 to 589 of SEQ ID NO:11). The human TANGO 228
cytoplasmic domain (SEQ ID NO:10) is represented by amino acids 250
to 343 (and encoded by nucleotides 781 to 1065 of SEQ ID NO:1), and
the rat MCA-32 cytoplasmic domain is represented by amino acids 200
to 278 (and encoded by nucleotides 590 to 829 of SEQ ID NO:11).
FIGS. 3A 3H and FIGS. 4A 4C show that there is an overall 28.3%
identity between the full length human TANGO 228 nucleic acid
molecule and the full length rat MCA-32 nucleic acid molecule, and
an overall 45.5% identity between the open reading frame of human
TANGO 228 nucleic acid molecule and the open reading frame of the
rat MCA-32 nucleic acid molecule, respectively. The amino acid
alignment in FIG. 5 shows a 26.8% overall amino acid sequence
identity between human TANGO 228 and rat MCA-32.
Clone EpT228, which encodes human TANGO 228, was deposited with the
American Type Culture Collection (10801 University Boulevard,
Manassas, Va. 20110-2209) on Feb. 18, 1999 and assigned Accession
Number 207116. This deposit will be maintained under the terms of
the Budapest Treaty on the International Recognition of the Deposit
of Microorganisms for the Purposes of Patent Procedure. This
deposit was made merely as a convenience for those of skill in the
art and is not an admission that a deposit is required under 35
U.S.C. .sctn.112.
FIG. 2 depicts a hydropathy plot of human TANGO 228. Relatively
hydrophobic regions of the protein are shown above the horizontal
line, and relatively hydrophilic regions of the protein are below
the horizontal line. The cysteine residues (cys) and
N-glycosylation sites are indicated by short vertical lines just
below the hydropathy trace. The dashed vertical line separates the
signal sequence (amino acids 1 to 19 of SEQ ID NO:3; SEQ ID NO:5)
on the left from the mature protein (amino acids 20 to 343 of SEQ
ID NO:3; SEQ ID NO:4) on the right. The TANGO 228 transmembrane
domain is indicated by the section of the plot under which the
number 7.1 can be seen, which represents a score assigned to the
predicted transmembrane domain. The extracellular domain (SEQ ID
NO:6) and cytoplasmic domain (SEQ ID NO:10) are similarly indicated
by gray horizontal bars, labeled as "out" and "in",
respectively.
Human TANGO 228 was mapped to h17q23. The nearby flanking markers
include: WI-4118 and WI-5110. The known nearby loci include: JPD
(periodontitis, juvenile) and DGI1 (dentinogenesis imperfecta).
Nearby known human genes include: COIL (coilin P80), TNFAIP1 (tumor
necrosis factor), GH1 (growth hormone1, 2), ICAM2 (intercellular
adhesion molecule), APOH (apolipoprotein H), PEPE (peptidase E),
MPO (myeloperoxidase), and CDK3 (cyclin dependent kinase3). Thus,
human TANGO 228 nucleic acid molecules can be used to map these
loci and genes.
The syntenic mouse chromosomal location is mo11. Nearby mouse loci
include: rimy (rimy), al (alopecia), nog (noggin), and bda
(baldarthritic). Near known mouse genes include: rimy (rimy), al
(alopecia), hlf (hepatic leukemia factor), chad (ahondroadherin),
re (rex), mpo (myeloperoxidase), nog (noggin), bda (bald
arthritic), gip (gastric inhibitory polypeptide), and ngfr (nerve
growth factor receptor).
Murine TANGO 228
A cDNA encoding murine TANGO 228 was identified by analyzing the
sequences of clones present in a bone marrow stromal cell cDNA
library. This analysis led to the identification of a clone,
jtmMa107f05, encoding full-length murine TANGO 228. The murine
TANGO 228 cDNA of this clone is 911 nucleotides long (FIG. 14; SEQ
ID NO:31). The open reading frame of this cDNA, nucleotides 27 to
743 of SEQ ID NO:31 (SEQ ID NO:32), encodes a 239 amino acid
transmembrane protein (FIG. 14; SEQ ID NO:33).
The signal peptide prediction program SIGNALP (Nielsen et al.
(1997) Protein Engineering 10:1 6) predicted that murine TANGO 228
includes a 34 amino acid signal peptide (amino acid 1 to about
amino acid 34 of SEQ ID NO:33; SEQ ID NO:35) preceding the mature
murine TANGO 228 protein (corresponding to about amino acid 35 to
amino acid 239 of SEQ ID NO:33; SEQ ID NO:34). The murine TANGO 228
protein molecular weight is 26.7 kDa prior to the cleavage of the
signal peptide, 22.9 kDa after cleavage of the signal peptide.
Mature murine TANGO 228 includes an extracellular domain (about
amino acids 35 to 141 of SEQ ID NO:33; SEQ ID NO:36), a
transmembrane domain (about amino acids 142 to 164 of SEQ ID NO:33;
SEQ ID NO:37), and a cytoplasmic domain (about amino acids 165 to
239 of SEQ ID NO:33; SEQ ID NO:8). Murine TANGO 228 also includes
an Ig domain (about amino acids 59 to 115 of SEQ ID NO:33; SEQ ID
NO:46).
FIGS. 15A 15B show an alignment of the murine TANGO 228 full length
nucleic acid sequence (upper line of each pair; SEQ ID NO:31) with
the human TANGO 228 full length nucleic acid sequence (lower line
of each pair; SEQ ID NO:1). FIGS. 16A 16B show an alignment of the
murine TANGO 228 coding region (upper line of each pair; SEQ ID
NO:32) with the human TANGO 228 coding region (lower line of each
pair; SEQ ID NO:2). FIG. 17 shows an alignment of the murine TANGO
228 protein sequence (upper line of each pair; SEQ ID NO:33) with
the human TANGO 228 protein sequence (lower line of each pair; SEQ
ID NO:3).
FIGS. 15A 15B and FIGS. 16A 4B show that there is an overall 58.2%
identity between the full length murine TANGO 228 nucleic acid
molecule and the full length human TANGO 228 nucleic acid molecule
over the length of the murine molecule and an overall 58.2%
identity between the open reading frame of murine TANGO 228 nucleic
acid molecule and the open reading frame of the human TANGO 228
nucleic acid molecule over the length of the murine molecule,
respectively. The amino acid alignment in FIG. 17 shows a 30.6%
overall amino acid sequence identity between murine TANGO 228 and
human TANGO 228 over the length of the murine moleceule
Uses of TANGO 228 Nucleic Acids, Polypeptides, and Modulators
Thereof
As TANGO 228 was originally found in a fetal spleen library, TANGO
228 nucleic acids, proteins, and modulators thereof can be used to
modulate the proliferation, differentiation, and/or function of
cells that form the spleen, e.g., cells of the splenic connective
tissue, e.g., splenic smooth muscle cells and/or endothelial cells
of the splenic blood vessels. TANGO 228 nucleic acids, proteins,
and modulators thereof can also be used to modulate the
proliferation, differentiation, and/or function of cells that are
processed, e.g., regenerated or phagocytized within the spleen,
e.g., erythrocytes and/or B and T lymphocytes and macrophages. Thus
TANGO 228 nucleic acids, proteins, and modulators thereof can be
used to treat spleen, e.g., the fetal spleen, associated diseases
and disorders. Examples of splenic diseases and disorders include
e.g., splenic lymphoma and/or splenomegaly, and/or phagocytotic
disorders, e.g., those inhibiting macrophage engulfment of bacteria
and viruses in the bloodstream.
Due to TANGO 228's homology to rat MCA-32, which is a cell surface
antigen gene up-regulated in activated mast cells, TANGO 228
nucleic acids, proteins, and modulators thereof can be used to
modulate mast cell function and thus to treat immunologic diseases
and disorders. Examples of immunologic diseases and disorders
include allergic disorders, e.g., anaphylaxis and allergic asthma,
and inflammatory disorders, e.g., atopic dermatitis.
Because TANGO 228 is homologous to rat MCA-32, which is expressed
in monocyte and macrophage cell lines, TANGO 228 nucleic acids,
proteins, and modulators thereof can be used to protect the body
from antigenic invaders, e.g., by modulating the activity of
macrophages, and can be used to treat allergies, e.g., anaphylactic
shock and/or allergic rhinitis.
In addition, as TANGO 228 includes one or more Ig domains, TANGO
228 nucleic acids, proteins, and modulators thereof can be used to
modulate immunologic function, e.g., by the modulation of
immunoglobulins and the formation of antibodies. For the same
reason, TANGO 228 nucleic acids, proteins, and modulators thereof
can be used to modulate Type I immunologic disorders, e.g.,
anaphylaxis and/or rhinitis, by modulating, e.g., stabilizing, the
interaction between antigens and mast cell receptors, e.g., high
affinity IgE receptors.
Due to its mapping in the same region as TNFAIP1, a tumor necrosis
factor, TANGO 228 nucleic acids, proteins, and modulators thereof
can be sued to treat TNF related disorders (e.g., acute
myocarditis, myocardial infarction, congestive heart failure, T
cell disorders (e.g., dermatitis, fibrosis)), differentiative and
apoptotic disorders, and disorders related to angiogenesis (e.g.,
tumor formation and/or metastasis, cancer). Modulators of TANGO 228
expression and/or activity can be used to treat such disorders.
Human TANGO 240
A cDNA encoding human TANGO 240 was identified by analyzing the
sequences of clones present in an osteoblast cDNA library. This
analysis led to the identification of a clone, jthoc087d01,
encoding full-length human TANGO 240. The human TANGO 240 cDNA of
this clone is 2165 nucleotides long (FIG. 6; SEQ ID NO:13). The
open reading frame of this cDNA, nucleotides 2 to 1126 of SEQ ID
NO:13 (SEQ ID NO:14), encodes a 374 amino acid secreted protein
(FIG. 6; SEQ ID NO:15).
The signal peptide prediction program SIGNALP (Nielsen, et al.
(1997) Protein Engineering 10:1 6) predicted that human TANGO 240
includes an 34 amino acid signal peptide (amino acid 1 to about
amino acid 34 of SEQ ID NO:15)(SEQ ID NO:17) preceding the mature
TANGO 240 protein (corresponding to about amino acid 35 to amino
acid 374 of SEQ ID NO:15; SEQ ID NO:16). The TANGO 240 protein
molecular weight is 40.6 kDa prior to the cleavage of the signal
peptide, 37.2 kDa after cleavage of the signal peptide.
An N-glycosylation site having the sequence NSTG is found from
amino acids 141 to 144 of SEQ ID NO:15. A cAMP and cGMP-dependent
protein kinase phosphorylation site having the sequence RRVT is
found from amino acids 117 to 120. A protein kinase C
phosphorylation site having the sequence SCR is found from amino
acids 234 to 236. A second protein kinase C phosphorylation site
having the sequence SGK is found from amino acids 323 to 325. A
casein kinase II phosphorylation site having the sequence SNTE is
found from amino acids 132 to 135. A second casein kinase II
phosphorylation site having the sequence TEAE is found from amino
acids 147 to 150. A third casein kinase II phosphorylation site
having the sequence SWND is found from amino acids 210 to 213. A
fourth casein kinase II phosphorylation site having the sequence
TEAE is found from amino acids 227 to 230. A fifth casein kinase II
phosphorylation site having the sequence TGED is found from amino
acids 270 to 273. A sixth casein kinase II phosphorylation site
having the sequence SVEE is found from amino acids 311 to 314. A
seventh casein kinase II phosphorylation site having the sequence
SGKD is found from amino acids 323 to 326. A tyrosine kinase
phosphorylation site having the sequence RVTIDAFY is found from
amino acids 118 to 125. An N-myristoylation site having the
sequence GLVCGR is found from amino acids 7 to 12. A second
N-myristoylation site having the sequence GAAGSQ is found from
amino acids 30 to 35. A third N-myristoylation site having the
sequence GTGAGA is found from amino acids 38 to 43. A fourth
N-myristoylation site having the sequence GSLAGS is found from
amino acids 44 to 49. A fifth N-myristoylation site having the
sequence GAHGNS is found from amino acids 59 to 64. A sixth
N-myristoylation site having the sequence GGLHNR is found from
amino acids 237 to 242. A seventh N-myristoylation site having the
sequence GQHYAN is found from amino acids 254 to 259. An eighth
N-myristoylation site having the sequence GSYMCH is found from
amino acids 332 to 337. An amidation site having the sequence AGKR
is found from amino acids 221 to 224. A prokaryotic membrane
lipoprotein lipid attachment site having the sequence GAGAGSLAGSC
is found from amino acids 40 to 50.
Clone EpT240, which encodes human TANGO 240, was deposited with the
American Type Culture Collection (10801 University Boulevard,
Manassas, Va. 20110-2209) on Feb. 18, 1999 and assigned Accession
Number 207116. This deposit will be maintained under the terms of
the Budapest Treaty on the International Recognition of the Deposit
of Microorganisms for the Purposes of Patent Procedure. This
deposit was made merely as a convenience for those of skill in the
art and is not an admission that a deposit is required under 35
U.S.C. .sctn.112.
FIG. 7 depicts a hydropathy plot of human TANGO 240. Relatively
hydrophobic regions of the protein are shown above the horizontal
line, and relatively hydrophilic regions of the protein are below
the horizontal line. The cysteine residues (cys) and potential
N-glycosylation sites (Ngly) are indicated by short vertical lines
just below the hydropathy trace. The dashed vertical line separates
the signal sequence (amino acids 1 to 34 of SEQ ID NO:15; SEQ ID
NO:17) on the left from the mature protein (amino acids 35 to 374
of SEQ ID NO:15; SEQ ID NO:16) on the right.
Northern analysis of TANGO 240 expression in human tissues showed
that an approximately 2.2 kB transcript is weakly expressed in
skeletal muscle, brain, colon, thymus, spleen, and small intestine,
moderately expressed in heart, liver, lung, and peripheral blood
leukocytes, and strongly expressed in kidney and placenta.
Secretion assays indicate that the polypeptide encoded by human
TANGO 240 is a secreted protein, as the presence of a 48 kD and a
42 kD protein was detected by performing the assays. The secretion
assays were performed essentially as follows: 8.times.10.sup.5 293T
cells were plated per well in a 6-well plate and the cells were
incubated in growth medium (DMEM, 10% fetal bovine serum,
penicillin/streptomycin) at 37.degree. C., 5% CO.sub.2 overnight.
293T cells were transfected with 2 g of full-length TANGO 240
inserted in the pMET7 vector/well and 10 g LipofectAMINE (GIBCO/BRL
Cat. #18324-012)/well according to the protocol for GIBCO/BRL
LipofectAMINE. The transfectant was removed 5 hours later and fresh
growth medium was added to allow the cells to recover overnight.
The medium was removed and each well was gently washed twice with
DMEM without methionine and cysteine (ICN Cat. #16-424-54). Next, 1
ml DMEM without methionine and cysteine with 50 Ci Trans-.sup.35S
(ICN Cat. #51006) was added to each well and the cells were
incubated at 37.degree. C., 5% CO.sub.2 for the appropriate time
period. A 150 l aliquot of conditioned medium was obtained and 150
l of 2.times.SDS sample buffer was added to the aliquot. The sample
was heat-activated and loaded on a 4 20% SDS-PAGE gel. The gel was
fixed and the presence of secreted protein was detected by
autoradiography.
Murine TANGO 240
A cDNA encoding murine TANGO 240 was identified by analyzing the
sequences of clones present in a mouse bone marrow stromal cell
cDNA library. This analysis led to the identification of a clone,
jtmMa100b11, encoding full-length murine TANGO 240. The murine
TANGO 240 cDNA of this clone is 2426 nucleotides long (FIGS. 18A
18B; SEQ ID NO:39). The open reading frame of this cDNA,
nucleotides 2 to 1117 of SEQ ID NO:39 (SEQ ID NO:40), encodes a 372
amino acid secreted protein (FIGS. 18A 18B; SEQ ID NO:41).
The signal peptide prediction program SIGNALP (Nielsen, et al.
(1997) Protein Engineering 10:1 6) predicted that murine TANGO 240
includes a 31 amino acid signal peptide (amino acid 1 to about
amino acid 31 of SEQ ID NO:41; SEQ ID NO:43) preceding the mature
murine TANGO 240 protein (corresponding to about amino acid 32 to
amino acid 372 of SEQ ID NO:11; SEQ ID NO:42). The murine TANGO 240
protein molecular weight is 40.7 kDa prior to the cleavage of the
signal peptide, 37.3 kDa after cleavage of the signal peptide.
FIGS. 19A 19D show an alignment of the murine TANGO 240 full length
nucleic acid sequence (upper line of each pair; SEQ ID NO:39) with
the human TANGO--240 full length nucleic acid sequence (lower line
of each pair; SEQ ID NO:13). FIGS. 20A 20B show an alignment of the
murine TANGO 240 coding region (upper line of each pair; SEQ ID
NO:40) with the human TANGO 240 coding region (lower line of each
pair; SEQ ID NO:14). FIG. 21 shows an alignment of the murine TANGO
240 protein sequence (upper line of each pair; SEQ ID NO:41) with
the human TANGO 240 protein sequence (lower line of each pair; SEQ
ID NO:15).
FIGS. 19A 19D and FIGS. 20A 20B show that there is an overall 78.4%
identity between the full length murine TANGO 240 nucleic acid
molecule and the full length human TANGO 240 nucleic acid molecule,
over the length of the human molecule, and an overall 84.4%
identity between the open reading frame of murine TANGO 240 nucleic
acid molecule and the open reading frame of the human TANGO 240
nucleic acid molecule, respectively. The amino acid alignment in
FIG. 21 shows an 86% overall amino acid sequence identity between
murine TANGO 240 and human TANGO 240.
Northern analysis of TANGO 240 expression in mouse tissues showed
that an approximately 2.2 kB transcript is weakly expressed in
spleen and skeletal muscle, moderately expressed in testis, and
strongly expressed in heart, brain, lung, liver, and kidney.
Uses of TANGO 240 Nucleic Acids, Polypeptides, and Modulators
Thereof
As TANGO 240 was originally found in an osteoblast library, TANGO
240 nucleic acids, proteins, and modulators thereof can be used to
modulate the proliferation, differentiation, and/or function of
bone and cartilage cells, e.g., chondrocytes and osteoblasts, and
to treat bone and/or cartilage associated diseases or disorders.
Examples of bone and/or cartilage diseases and disorders include
bone and/or cartilage injury due to for example, trauma (e.g., bone
breakage, cartilage tearing), degeneration (e.g., osteoporosis),
degeneration of joints, e.g., arthritis, e.g., osteoarthritis, and
bone wearing.
As TANGO 240 is homologous to Mycobacterium tuberculosis conserved
hypothetical protein Rv0712, TANGO 240 nucleic acids, proteins, and
modulators thereof can be used to treat diseases associated with
bacterial infection, e.g., tuberculosis, e.g., pulmonary
tuberculosis. In addition, TANGO 240 can be used to modulate, e.g.,
trigger, the immune response and can be used to treat immunologic
disease and disorders, e.g., those associated with the respiratory
system, e.g., asthma.
TANGO 240 nucleic acids, proteins, and modulators thereof can also
be used to treat disorders of the cells and tissues in which it is
expressed. As TANGO 240 is expressed in heart, brain, spleen, lung,
liver, skeletal muscle, kidney, testis, colon, thymus, peripheral
blood leukocytes, small intestine, and placenta, TANGO 240 nucleic
acids, proteins, and modulators thereof can be used to treat
disorders of these cells, tissues, or organs, e.g., ischemic heart
disease or atherosclerosis, head trauma, brain cancer, splenic
lymphoma, splenomegaly, lung cancer, cystic fibrosis, rheumatoid
lung disease, liver cirrhosis, hepatitis, muscular dystrophy,
stroke, muscular atrophy, glomerulonephritis, end stage renal
disease, uremia, testicular cancer, colon cancer and colonic
volvulus, DiGeorge syndrome, thymoma, autoimmune disorders,
atresia, Crohns disease, and various placental disorders.
Human TANGO 243
A cDNA encoding human TANGO 243 was identified by analyzing the
sequences of clones present in a fetal spleen cDNA library. This
analysis led to the identification of a clone, jthsa049g04,
encoding potentially full-length human TANGO 243. The human TANGO
243 cDNA of this clone is 2811 nucleotides long (FIG. 9; SEQ ID
NO:19). The open reading frame of this cDNA, nucleotides 183 to
2567 of SEQ ID NO:19 (SEQ ID NO:20), encodes a 795 amino acid
protein (FIG. 9; SEQ ID NO:21). The TANGO 243 protein molecular
weight is 87.1 kDa.
Like TANGO 243, proteins with a WD domain contain a number of
conserved G-beta repeats and are intracellular. The functions
associated with WD domains and G-beta repeats, e.g., signal
transduction, cell division control, transcriptional regulation,
vesicular trafficking, are performed within the cell.
TANGO 243 includes 6 G-beta domains and one G-beta-like domain.
G-beta domain 1 is located at about amino acids 8 to 47 of SEQ ID
NO:21 (SEQ ID NO:22). G-beta domain 2 is located at about amino
acids 55 to 98 of SEQ ID NO:21 (SEQ ID NO:23). G-beta domain 3 is
located at about amino acids 102 to 139 of SEQ ID NO:21 (SEQ ID
NO:24). G-beta domain 4 is located at about amino acids 141 to 179
of SEQ ID NO:21 (SEQ ID NO:25). G-beta domain 5 is located at about
amino acids 181 to 218 of SEQ ID NO:21 (SEQ ID NO:26). G-beta
domain 6 is located at about amino acids 221 to 259 of SEQ ID NO:21
(SEQ ID NO:27). The G-beta-like domain is located at about amino
acids 261 to 298 of SEQ ID NO:21 (SEQ ID NO:28).
An N-glycosylation site having the sequence NRSF is found from
amino acids 52 to 55 of SEQ ID NO:21. A second N-glycosylation site
having the sequence NTSD is found from amino acids 421 to 424. A
third N-glycosylation site having the sequence NGTA is found from
amino acids 559 to 562. A fourth N-glycosylation site having the
sequence NSSS is found from amino acids 585 to 588. A fifth
N-glycosylation site having the sequence NYSV is found from amino
acids 708 to 711. A cAMP and cGMP-dependent protein kinase
phosphorylation site having the sequence KKLT is found from amino
acids 566 to 569. A second cAMP and cGMP-dependent protein kinase
phosphorylation site having the sequence KKYS is found from amino
acids 773 to 776. A protein kinase C phosphorylation site having
the sequence SLR is found from amino acids 13 to 15. A second
protein kinase C phosphorylation site having the sequence TTR is
found from amino acids 42 to 44. A third protein kinase C
phosphorylation site having the sequence SGK is found from amino
acids 120 to 122. A fourth protein kinase C phosphorylation site
having the sequence TAK is found from amino acids 134 to 136. A
fifth protein kinase C phosphorylation site having the sequence TVK
is found from amino acids 174 to 176. A sixth protein kinase C
phosphorylation site having the sequence SIR is found from amino
acids 213 to 215. A seventh protein kinase C phosphorylation site
having the sequence SLR is found from amino acids 254 to 256. An
eighth protein kinase C phosphorylation site having the sequence
TIR is found from amino acids 266 to 268. A ninth protein kinase C
phosphorylation site having the sequence SGK is found from amino
acids 391 to 393. A tenth protein kinase C phosphorylation site
having the sequence SYK is found from amino acids 415 to 417. An
eleventh protein kinase C phosphorylation site having the sequence
SEK is found from amino acids 588 to 590. A twelfth protein kinase
C phosphorylation site having the sequence SIK is found from amino
acids 620 to 622. A thirteenth protein kinase C phosphorylation
site having the sequence SQR is found from amino acids 678 to 680.
A fourteenth protein kinase C phosphorylation site having the
sequence SNK is found from amino acids 694 to 696. A fifteenth
protein kinase C phosphorylation site having the sequence TFR is
found from amino acids 742 to 744. A casein kinase II
phosphorylation site having the sequence SFTE is found from amino
acids 54 to 57. A second casein kinase II phosphorylation site
having the sequence SGHE is found from amino acids 188 to 191. A
third casein kinase II phosphorylation site having the sequence
SETE is found from amino acids 201 to 204. A fourth casein kinase
II phosphorylation site having the sequence TTAE is found from
amino acids 248 to 251. A fifth casein kinase II phosphorylation
site having the sequence TESE is found from amino acids 298 to 301.
A sixth casein kinase II phosphorylation site having the sequence
SAEE is found from amino acids 306 to 309. A seventh casein kinase
II phosphorylation site having the sequence SVSE is found from
amino acids 368 to 371. An eighth casein kinase II phosphorylation
site having the sequence TSDD is found from amino acids 422 to 425.
A ninth casein kinase II phosphorylation site having the sequence
SFSD is found from amino acids 466 to 469. A tenth casein kinase II
phosphorylation site having the sequence TAPE is found from amino
acids 561 to 564. An eleventh casein kinase II phosphorylation site
having the sequence TEDD is found from amino acids 569 to 572. A
twelfth casein kinase II phosphorylation site having the sequence
SSSE is found from amino acids 586 to 589. A thirteenth casein
kinase II phosphorylation site having the sequence SVNE is found
from amino acids 625 to 628. A fourteenth casein kinase II
phosphorylation site having the sequence SQRE is found from amino
acids 678 to 681. A fifteenth casein kinase II phosphorylation site
having the sequence TILE is found from amino acids 731 to 734. A
sixteenth casein kinase II phosphorylation site having the sequence
SVSE is found from amino acids 777 to 780. An N-myristoylation site
having the sequence GLVCCA is found from amino acids 23 to 28. A
second N-myristoylation site having the sequence GAFVSV is found
from amino acids 32 to 37. A third N-myristoylation site having the
sequence GLIATG is found from amino acids 83 to 88. A fourth
N-myristoylation site having the sequence GNDHNI is found from
amino acids 89 to 94. A fifth N-myristoylation site having the
sequence GTLLSG is found from amino acids 124 to 129. A sixth
N-myristoylation site having the sequence GLMLTG is found from
amino acids 164 to 169. A seventh N-myristoylation site having the
sequence GASDGI is found from amino acids 288 to 293. An eighth
N-myristoylation site having the sequence GTREGQ is found from
amino acids 347 to 352. A ninth N-myristoylation site having the
sequence GSSGAN is found from amino acids 382 to 387. A tenth
N-myristoylation site having the sequence GQMLGL is found from
amino acids 457 to 462. An eleventh N-myristoylation site having
the sequence GSSGSS is found from amino acids 480 to 485. A twelfth
N-myristoylation site having the sequence GTTMAG is found from
amino acids 508 to 513. A thirteenth N-myristoylation site having
the sequence GAQFSS is found from amino acids 636 to 641. A
fourteenth N-myristoylation site having the sequence GSNKNI is
found from amino acids 693 to 698. A fifteenth N-myristoylation
site having the sequence GTLISD is found from amino acids 750 to
755.
Clone EpT243, which encodes human TANGO 243, was deposited with the
American Type Culture Collection (10801 University Boulevard,
Manassas, Va. 20110 2209) on Feb. 18, 1999 and assigned Accession
Number 207116. This deposit will be maintained under the terms of
the Budapest Treaty on the International Recognition of the Deposit
of Microorganisms for the Purposes of Patent Procedure. This
deposit was made merely as a convenience for those of skill in the
art and is not an admission that a deposit is required under 35
U.S.C. .sctn.112.
FIG. 10 depicts a hydropathy plot of human TANGO 243. Relatively
hydrophobic regions of the protein are shown above the horizontal
line, and relatively hydrophilic regions of the protein are below
the horizontal line. The cysteine residues (cys) and potential
N-glycosylation sites (Ngly) are indicated by short vertical lines
just below the hydropathytrace.
TANGO 243 bears some similarity to human PLAP (GenBank Accession
Number AF083395). An alignment of the nucleotide sequence of human
PLAP (SEQ ID NO:29; GenBank Accession Number AF083395) and the
nucleotide sequence of human TANGO 243 (SEQ ID NO:19) is depicted
in FIGS. 11A 11F. In this alignment, the nucleotide sequences of
human PLAP and human TANGO 243 are 78.8% identical. This alignment
was performed using the ALIGN alignment program with a PAM120
scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
FIGS. 12A 12E depict an alignment of the nucleotide sequence of the
open reading frames of human PLAP (nucleotides 1 to 2217 of SEQ ID
NO:29) and human TANGO 243 (SEQ ID NO:20). The nucleotide sequences
of the open reading frames of human PLAP and human TANGO 243 (SEQ
ID NO:20) are 92.7% identical. This alignment was performed using
the ALIGN alignment program with a PAM120 scoring matrix, a gap
length penalty of 12, and a gap penalty of 4. An alignment of the
amino acid sequence of human PLAP (SEQ ID NO:30) and the amino acid
sequence of human TANGO 243 (SEQ ID NO:21) is depicted in FIGS. 13A
13B. The amino acid sequences of human PLAP and human TANGO 243 are
92.8% identical. This alignment was performed using the ALIGN
alignment program with a PAM120 scoring matrix, a gap length
penalty of 12, and a gap penalty of 4.
It is possible that the human TANGO 243 clone described above,
jthsa049g04, is a less than full-length clone and encodes a portion
of human TANGO 243. If so, the start site of translation is located
upstream of the 5' end of this clone. FIG. 22 depicts an
alternative translation of the 2811 nucleotide long human TANGO 243
cDNA clone described above. In this translation, the open reading
frame of the cDNA, nucleotides 3 to 2567 of SEQ ID NO: 19 (SEQ ID
NO: 44), encodes a 855 amino acid protein (FIG. 22; SEQ ID NO: 45).
The molecular weight of this TANGO 243 protein is 93.7 kDa.
In this alternative translation of the human TANGO 243, G-beta
domain 1 is located at about amino acids 68 to 107 of SEQ ID NO:45
(SEQ ID NO:22), G-beta domain 2 is located at about amino acids 115
to 158 of SEQ ID NO:45 (SEQ ID NO:23), G-beta domain 3 is located
at about amino acids 162 to 199 of SEQ ID NO:45 (SEQ ID NO:24),
G-beta domain 4 is located at about amino acids 201 to 239 of SEQ
ID NO:45 (SEQ ID NO:25), G-beta domain 5 is located at about amino
acids 241 to 278 of SEQ ID NO:45 (SEQ ID NO:26), G-beta domain 6 is
located at about amino acids 281 to 319 of SEQ ID NO:45 (SEQ ID
NO:27), and the G-beta-like domain is located at about amino acids
321 to 358 of SEQ ID NO:45 (SEQ ID NO:28).
Within this longer form of TANGO 243, there are regions with
considerable similarity to human PLAP. For example, a region of
human TANGO 243 from amino acids 358 to 609 of SEQ ID NO:45 (SEQ ID
NO:47) constitute PLAP-like domain 1. A region of human TANGO 243
from amino acids 528 to 758 of SEQ ID NO:45 (SEQ ID NO:48)
constitute PLAP-like domain 2. A region of human TANGO 243 from
amino acids 118 to 205 of SEQ ID NO:45 (SEQ ID NO:49) constitute
PLAP-like domain 3. A region of human TANGO 243 from amino acids
239 to 273 of SEQ ID NO:45 (SEQ ID NO:50) constitute PLAP-like
domain 4.
In this alternative translation of the TANGO 243 clone the
following sites are present. An N-glycosylation site having the
sequence NRSF is found from amino acids 112 to 115 of SEQ ID NO:45.
A second N-glycosylation site having the sequence NTSD is found
from amino acids 481 to 484. A third N-glycosylation site having
the sequence NGTA is found from amino acids 619 to 622. A fourth
N-glycosylation site having the sequence NSSS is found from amino
acids 645 to 648. A fifth N-glycosylation site having the sequence
NYSV is found from amino acids 768 to 771. A cAMP and
cGMP-dependent protein kinase phosphorylation site having the
sequence KKLT is found from amino acids 626 to 629. A second cAMP
and cGMP-dependent protein kinase phosphorylation site having the
sequence KKYS is found from amino acids 833 to 836. A protein
kinase C phosphorlyation site having the sequence SGR is found from
amino acids 22 to 24. A second protein kinase C phosphorylation
site having the sequence SLR is found from amino acids 73 to 75. A
third protein kinase C phosphorylation site having the sequence TTR
is found from amino acids 102 to 104. A fourth protein kinase C
phosphorylation site having the sequence SGK is found from amino
acids 180 to 182. A fifth protein kinase C phosphorylation site
having the sequence TAK is found from amino acids 194 to 196. A
sixth protein kinase C phosphorylation site having the sequence TVK
is found from amino acids 234 to 236. A seventh protein kinase C
phosphorylation site having the sequence SIR is found from amino
acids 273 to 275. An eighth protein kinase C phosphorylation site
having the sequence SLR is found from amino acids 314 to 316. A
ninth protein kinase C phosphorylation site having the sequence TIR
is found from amino acids 326 to 328. A tenth protein kinase C
phosphorylation site having the sequence SGK is found from amino
acids 451 to 453. An eleventh protein kinase C phosphorylation site
having the sequence SYK is found from amino acids 475 to 477. A
twelfth protein kinase C phosphorylation site having the sequence
SEK is found from amino acids 648 to 650. A thirteenth protein
kinase C phosphorylation site having the sequence SIK is found from
amino acids 680 to 682. A fourteenth protein kinase C
phosphorylation site having the sequence SQR is found from amino
acids 738 to 740. A fifteenth protein kinase C phosphorylation site
having the sequence SNK is found from amino acids 754 to 756. A
sixteenth protein kinase C phosphorylation site having the sequence
TFR is found from amino acids 802 to 804. A casein kinase II
phosphorylation site having the sequence SFTE is found from amino
acids 114 to 117. A second casein kinase II phosphorylation site
having the sequence SGHE is found from amino acids 248 to 252. A
third casein kinase II phosphorylation site having the sequence
SETE is found from amino acids 261 to 264. A fourth casein kinase
II phosphorylation site having the sequence TTAE is found from
amino acids 308 to 331. A fifth casein kinase II phosphorylation
site having the sequence TESE is found from amino acids 358 to 361.
A sixth casein kinase II phosphorylation site having the sequence
SAEE is found from amino acids 366 to 369. A seventh casein kinase
II phosphorylation site having the sequence SVSE is found from
amino acids 428 to 431. An eighth casein kinase II phosphorylation
site having the sequence TSDD is found from amino acids 482 to 485.
A ninth casein kinase II phosphorylation site having the sequence
SFSD is found from amino acids 526 to 529. A tenth casein kinase II
phosphorylation site having the sequence TAPE is found from amino
acids 621 to 624. An eleventh casein kinase II phosphorylation site
having the sequence TEDD is found from amino acids 629 to 632. A
twelfth casein kinase II phosphorylation site having the sequence
SSSE is found from amino acids 646 to 649. A thirteenth casein
kinase II phosphorylation site having the sequence SVNE is found
from amino acids 685 to 688. A fourteenth casein kinase II
phosphorylation site having the sequence SQRE is found from amino
acids 738 to 741. A fifteenth casein kinase II phosphorylation site
having the sequence TILE is found from amino acids 791 to 794. A
sixteenth casein kinase II phosphorylation site having the sequence
SVSE is found from amino acids 837 to 840. An N-myristoylation site
having the sequence GSSPAA is found from amino acids 29 to 34. A
second N-myristoylation site having the sequence GARQTL is found
from amino acids 54 to 59. A third N-myristoylation site having the
sequence GLVCCA is found from amino acids 83 to 88. A fourth
N-myristoylation site having the sequence GAFVSV is found from
amino acids 92 to 97. A fifth N-myristoylation site having the
sequence GLIATG is found from amino acids 83 to 88. A sixth
N-myristoylation site having the sequence GNDHNI is found from
amino acids 149 to 154. A seventh N-myristoylation site having the
sequence GTLLSG is found from amino acids 184 to 189. An eighth
N-myristoylation site having the sequence GLMLTG is found from
amino acids 224 to 229. A ninth N-myristoylation site having the
sequence GASDGI is found from amino acids 348 to 353. An tenth
N-myristoylation site having the sequence GTREGQ is found from
amino acids 407 to 412. A eleventh N-myristoylation site having the
sequence GSSGAN is found from amino acids 442 to 447. A twelfth
N-myristoylation site having the sequence GQMLGL is found from
amino acids 517 to 522. A thirteenth N-myristoylation site having
the sequence GSSGSS is found from amino acids 540 to 545. A
fourteenth N-myristoylation site having the sequence GTTMAG is
found from amino acids 568 to 573. A fifteenth N-myristoylation
site having the sequence GAQFSS is found from amino acids 696 to
701. A sixteenth N-myristoylation site having the sequence GSNKNI
is found from amino acids 753 to 758. A seventeenth
N-myristoylation site having the sequence GTLISD is found from
amino acids 810 to 815.
FIG. 23 depicts a hydropathy plot of the 855 amino acid human TANGO
243 protein arising from the alternative translation. Relatively
hydrophobic regions of the protein are shown above the horizontal
line, and relatively hydrophilic regions of the protein are below
the horizontal line. The cysteine residues (cys) and potential
N-glycosylation sites (Ngly) are indicated by short vertical lines
just below the hydropathy trace.
Uses of TANGO 243 Nucleic Acids, Polypeptides, and Modulators
Thereof
As TANGO 243 was originally found in a fetal spleen library, TANGO
243 nucleic acids, proteins, and modulators thereof can be used to
modulate the proliferation, differentiation, and/or function of
cells that form the spleen, e.g., cells of the splenic connective
tissue, e.g., splenic smooth muscle cells and/or endothelial cells
of the splenic blood vessels. TANGO 243 nucleic acids, proteins,
and modulators thereof can also be used to modulate the
proliferation, differentiation, and/or function of cells that are
processed, e.g., regenerated or phagocytized within the spleen,
e.g., erythrocytes and/or B and T lymphocytes and macrophages. Thus
TANGO 243 nucleic acids, proteins, and modulators thereof can be
used to treat spleen, e.g., the fetal spleen, associated diseases
and disorders. Examples of splenic diseases and disorders include
e.g., splenic lymphoma and/or splenomegaly, and/or phagocytotic
disorders, e.g., those inhibiting macrophage engulfment of bacteria
and viruses in the bloodstream.
Since TANGO 243 is homologous to human PLAP, which is up-regulated
in patients with rheumatoid arthritis, and which activates
molecules involved in the arthropathy pathway, TANGO 243 nucleic
acids, proteins, and modulators thereof can be used to treat
inflammatory arthropathy, and in bone and cartilage diseases and
disorders, e.g., bone and cartilage degenerative diseases and
disorders, e.g., arthritis, e.g., rheumatoid arthritis.
Because both TANGO 243 and PLAP hydrolyze phospholipids into
lysophospholipids, which are known to modulate chemotaxis, regulate
blood vessel permeability, and promote membrane fusion, and can
cause monocytes to secrete IL-1 (interleukin 1), in turn modulating
T-cell-mediated immunity, TANGO 243 nucleic acids, proteins and
modulators thereof can be used to modulate chemotaxis, regulate
blood vessel permeability, and promote membrane fusion, and can
cause monocytes to secrete IL-1 (interleukin 1), in turn modulating
T-cell-mediated immunity.
TANGO 243 and PLAP can also be used to modulate IL-1 and TNF (tumor
necrosis factor) synthesis and release (e.g., by modulating, e.g.,
activating PLA2 (phospholipase A2), which releases arachidonic acid
as a byproduct of hydrolysis, which regulates IL-1 and TNF
transcription). As IL-1 and TNF are involved in immunologic
processes, TANGO 243 nucleic acids, proteins, and modulators
thereof can be used to modulate such processes and to treat
immunologic disorders.
By activating PLA2, TANGO 243 nucleic acids, proteins, and
modulators thereof can modulate levels of arachidonic acid, and
thus control levels of eicosanoids and prostaglandins. By
controlling the levels of eicosanoids and prostaglandins, TANGO 243
nucleic acids, proteins, and modulators thereof can modulate the
immunologic pathway.
The presence of one or more G-beta domain suggests that TANGO 228
functions in a manner similar to other G-beta-containing proteins,
such as members of the WD-repeat family. For example, members of
WD-repeat family typically have biological functions that include,
but are not limited to, signal transduction, cell divisional
control, transcription regulation, and vesicular trafficking.
Tables 1 and 2 below provide a summary of the sequence information
for human TANGO 228, human TANGO 240, and human TANGO 243. Tables 3
and 4 below provide a summary of the sequence information for
murine TANGO 228 and murine TANGO 240.
TABLE-US-00001 TABLE 1 Summary of Human TANGO 228, TANGO 240 and
TANGO 243 Sequence Information Accession Gene cDNA ORF Figure
Number TANGO 228 SEQ ID SEQ ID FIGS. 1A 1C 207116 NO: 1 NO: 2 TANGO
240 SEQ ID SEQ ID FIGS. 6A 6B 207116 NO: 13 NO: 14 TANGO 243 SEQ ID
SEQ ID FIGS. 9A 9C 207116 NO: 19 NO: 20 TANGO 243 SEQ ID SEQ ID
FIGS. 22A 22C 207116 Alternative NO: 19 NO: 44 translation
TABLE-US-00002 TABLE 2 Summary of Domains of Human TANGO 228, TANGO
240, and TANGO 243 Proteins Signal Mature Protein Sequence Protein
Extracellular Ig Transmembrane Cytoplasmic G-beta- TANGO aa 1 19 aa
20 343 aa 1 227 aa 49 105 aa 228 249 aa 250 343 228 of SEQ ID of
SEQ ID of SEQ ID and of SEQ ID of SEQ ID NO: 3 NO: 3 NO: 3 140 198
NO: 3 NO: 3 (SEQ ID (SEQ ID (SEQ ID of SEQ ID (SEQ ID (SEQ ID NO:
5) NO: 4) NO: 6) NO: 3 NO: 7) NO: 10) (SEQ ID NO: 8 and 9) TANGO aa
1 34 aa 35 374 240 of SEQ ID of SEQ ID NO: 13 NO: 13 (SEQ ID (SEQ
ID NO: 17) NO: 16) TANGO aa 8 47, 243 55 98, 102 139, 141 179, 181
218, 221 259, and 261 298 of SEQ ID NO: 19 (SEQ ID NO: 22 28) TANGO
aa 68 107, 243 115 158, Alternative 162 199, Translation 201 239,
241 278, 281 319, and 321 358 of SEQ ID NO: 45 (SEQ ID NO: 22
28)
TABLE-US-00003 TABLE 3 Summary of Murine TANGO 228 and Murine TANGO
240 Sequence Information Accession Gene cDNA ORF Figure Number
TANGO 228 SEQ ID NO: 31 SEQ ID NO: 32 FIG. 14 TANGO 240 SEQ ID NO:
43 SEQ ID NO: 44 FIG. 18
TABLE-US-00004 TABLE 4 Summary of Domains of Murine TANGO 228 and
TANGO 240 Proteins Protein Signal Sequence Mature Protein
Extracellular Transmembrane Cytoplasmic TANGO aa 1 34 aa 35 239 aa
35 141 aa 142 164 aa 165 239 228 of SEQ ID of SEQ ID of SEQ ID of
SEQ ID of SEQ ID NO: 33 NO: 33 NO: 33 NO: 33 NO: 33 (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID NO: 35) NO: 34) NO: 36) NO: 37) NO: 38)
TANGO aa 1 31 aa 32 372 240 of SEQ ID of SEQ ID NO: 41 NO: 41 (SEQ
ID (SEQ ID NO: 43) NO: 42)
Various aspects of the invention are described in further detail in
the following subsections:
I. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid
molecules that encode a polypeptide of the invention or a
biologically active portion thereof, as well as nucleic acid
molecules sufficient for use as hybridization probes to identify
nucleic acid molecules encoding a polypeptide of the invention and
fragments of such nucleic acid molecules suitable for use as PCR
primers for the amplification or mutation of nucleic acid
molecules. As used herein, the term "nucleic acid molecule" is
intended to include DNA molecules (e.g., cDNA or genomic DNA) and
RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
An "isolated" nucleic acid molecule is one which is separated from
other nucleic acid molecules which are present in the natural
source of the nucleic acid molecule. Preferably, an "isolated"
nucleic acid molecule is free of sequences (preferably protein
encoding sequences) which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
For example, in various embodiments, the isolated nucleic acid
molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB,
0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the
nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived. Moreover, an "isolated" nucleic acid
molecule, such as a cDNA molecule, can be substantially free of
other cellular material, or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized. As used
herein, the term "isolated" when referring to a nucleic acid
molecule does not include an isolated chromosome.
A nucleic acid molecule of the present invention, e.g., a nucleic
acid molecule having the nucleotide sequence of SEQ ID NO:1, 2, 3,
13, 14, 19, 20, 31, 32, 39, 40, or 44, or a complement thereof, can
be isolated using standard molecular biology techniques and the
sequence information provided herein. Using all or a portion of the
nucleic acid sequences of SEQ ID NO:1, 2, 3, 13, 14, 19, 20, 31,
32, 39, 40, or 44 as a hybridization probe, nucleic acid molecules
of the invention can be isolated using standard hybridization and
cloning techniques (e.g., as described in Sambrook et al., eds.,
Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989).
A nucleic acid molecule of the invention can be amplified using
cDNA, mRNA or genomic DNA as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to all or a portion of
a nucleic acid molecule of the invention can be prepared by
standard synthetic techniques, e.g., using an automated DNA
synthesizer.
In another preferred embodiment, an isolated nucleic acid molecule
of the invention comprises a nucleic acid molecule which is a
complement of the nucleotide sequence of SEQ ID NO:1, 2, 3, 13, 14,
19, 20, 31, 32, 39, 40, or 44, or a portion thereof. A nucleic acid
molecule which is complementary to a given nucleotide sequence is
one which is sufficiently complementary to the given nucleotide
sequence that it can hybridize to the given nucleotide sequence
thereby forming a stable duplex.
Moreover, a nucleic acid molecule of the invention can comprise
only a portion of a nucleic acid sequence encoding a full length
polypeptide of the invention for example, a fragment which can be
used as a probe or primer or a fragment encoding a biologically
active portion of a polypeptide of the invention. The nucleotide
sequence determined from the cloning one gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning homologues in other cell types, e.g., from other
tissues, as well as homologues from other mammals. The probe/primer
typically comprises substantially purified oligonucleotide. The
oligonucleotide typically comprises a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 12,
preferably about 25, more preferably about 50, 75, 100, 125, 150,
175, 200, 250, 300, 350 or 400 consecutive nucleotides of the sense
or anti-sense sequence of SEQ ID NO:1, 2, 3, 13, 14, 19, 20, 31,
32, 39, 40, or 44 or of a naturally occurring mutant of SEQ ID
NO:1, 2, 3, 13, 14, 19, 20, 31, 32, 39, 40, or 44.
Probes based on the sequence of a nucleic acid molecule of the
invention can be used to detect transcripts or genomic sequences
encoding the same protein molecule encoded by a selected nucleic
acid molecule. The probe comprises a label group attached thereto,
e.g., a radioisotope, a fluorescent compound, an enzyme, or an
enzyme co-factor. Such probes can be used as part of a diagnostic
test kit for identifying cells or tissues which mis-express the
protein, such as by measuring levels of a nucleic acid molecule
encoding the protein in a sample of cells from a subject, e.g.,
detecting mRNA levels or determining whether a gene encoding the
protein has been mutated or deleted.
A nucleic acid fragment encoding a "biologically active portion" of
a polypeptide of the invention can be prepared by isolating a
portion of any of SEQ ID NO:2, 14, or 20, expressing the encoded
portion of the polypeptide protein (e.g., by recombinant expression
in vitro) and assessing the activity of the encoded portion of the
polypeptide.
The invention further encompasses nucleic acid molecules that
differ from the nucleotide sequence of SEQ ID NO:1, 2, 3, 13, 14,
19, 20, 31, 32, 39, 40, or 44, due to degeneracy of the genetic
code and thus encode the same protein as that encoded by the
nucleotide sequence of SEQ ID NO:2, 14, or 20.
In addition to the nucleotide sequences of SEQ ID NO:2, 14, and 20,
it will be appreciated by those skilled in the art that DNA
sequence polymorphisms that lead to changes in the amino acid
sequence may exist within a population (e.g., the human
population). Such genetic polymorphisms may exist among individuals
within a population due to natural allelic variation. An allele is
one of a group of genes which occur alternatively at a given
genetic locus. As used herein, the phrase "allelic variant" refers
to a nucleotide sequence which occurs at a given locus or to a
polypeptide encoded by the nucleotide sequence. As used herein, the
terms "gene" and "recombinant gene" refer to nucleic acid molecules
comprising an open reading frame encoding a polypeptide of the
invention. Such natural allelic variations can typically result in
1 5% variance in the nucleotide sequence of a given gene.
Alternative alleles can be identified by sequencing the gene of
interest in a number of different individuals. This can be readily
carried out by using hybridization probes to identify the same
genetic locus in a variety of individuals. Any and all such
nucleotide variations and resulting amino acid polymorphisms or
variations that are the result of natural allelic variation and
that do not alter the functional activity are intended to be within
the scope of the invention.
Moreover, nucleic acid molecules encoding proteins of the invention
from other species (homologues), which have a nucleotide sequence
which differs from that of the human protein described herein are
intended to be within the scope of the invention. Nucleic acid
molecules corresponding to natural allelic variants and homologues
of a cDNA of the invention can be isolated based on their identity
to the human nucleic acid molecule disclosed herein using the human
cDNAs, or a portion thereof, as a hybridization probe according to
standard hybridization techniques under stringent hybridization
conditions. For example, a cDNA encoding a soluble form of a
membrane-bound protein of the invention isolated based on its
hybridization to a nucleic acid molecule encoding all or part of
the membrane-bound form. Likewise, a cDNA encoding a membrane-bound
form can be isolated based on its hybridization to a nucleic acid
molecule encoding all or part of the soluble form.
Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention is at least 300 (325, 350, 375, 400, 425,
450, 500, 550, 600, 650, 700, 800, 900, or 1000) nucleotides in
length and hybridizes under stringent conditions to the nucleic
acid molecule comprising the nucleotide sequence, preferably the
coding sequence, of SEQ ID NO:1 or 13, or a complement thereof.
Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention is at least 600 (650, 700, 800, 900,
1000, 1200, 1400, 1600, 1800, 2000, or 2200) nucleotides in length
and hybridizes under stringent conditions to the nucleic acid
molecule comprising the nucleotide sequence, preferably the coding
sequence, of SEQ ID NO:19, or a complement thereof.
As used herein, the term "hybridizes under stringent conditions" is
intended to describe conditions for hybridization and washing under
which nucleotide sequences at least 60% (65%, 70%, preferably 75%)
identical to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1 6.3.6. A preferred, non-limiting
example of stringent hybridization conditions are hybridization in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at 50
65.degree. C. Preferably, an isolated nucleic acid molecule of the
invention that hybridizes under stringent conditions to the
sequence of SEQ ID NO:1, 2, 3, 13, 14, 19, 20, 31, 32, 39, 40, or
44, or a complement thereof, corresponds to a naturally-occurring
nucleic acid molecule. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein).
In addition to naturally-occurring allelic variants of a nucleic
acid molecule of the invention sequence that may exist in the
population, the skilled artisan will further appreciate that
changes can be introduced by mutation thereby leading to changes in
the amino acid sequence of the encoded protein, without altering
the biological activity of the protein. For example, one can make
nucleotide substitutions leading to amino acid substitutions at
"non-essential" amino acid residues. A "non-essential" amino acid
residue is a residue that can be altered from the wild-type
sequence without altering the biological activity, whereas an
"essential" amino acid residue is required for biological activity.
For example, amino acid residues that are not conserved or only
semi-conserved among homologues of various species may be
non-essential for activity and thus would be likely targets for
alteration. Alternatively, amino acid residues that are conserved
among the homologues of various species (e.g., murine and human)
may be essential for activity and thus would not be likely targets
for alteration.
Accordingly, another aspect of the invention pertains to nucleic
acid molecules encoding a polypeptide of the invention that contain
changes in amino acid residues that are not essential for activity.
Such polypeptides differ in amino acid sequence from SEQ ID NO:3,
yet retain biological activity. In one embodiment, the isolated
nucleic acid molecule includes a nucleotide sequence encoding a
protein that includes an amino acid sequence that is at least about
30% identical, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95%, or
98% identical to the amino acid sequence of SEQ ID NO:3.
Accordingly, another aspect of the invention pertains to nucleic
acid molecules encoding a polypeptide of the invention that contain
changes in amino acid residues that are not essential for activity.
Such polypeptides differ in amino acid sequence from SEQ ID NO:15,
yet retain biological activity. In one embodiment, the isolated
nucleic acid molecule includes a nucleotide sequence encoding a
protein that includes an amino acid sequence that is at least about
35% identical, 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%
identical to the amino acid sequence of SEQ ID NO:15.
Accordingly, another aspect of the invention pertains to nucleic
acid molecules encoding a polypeptide of the invention that contain
changes in amino acid residues that are not essential for activity.
Such polypeptides differ in amino acid sequence from SEQ ID NO:21,
yet retain biological activity. In one embodiment, the isolated
nucleic acid molecule includes a nucleotide sequence encoding a
protein that includes an amino acid sequence that is at least about
93% identical, 94%, 95%, 96%, 97% or 98% identical to the amino
acid sequence of SEQ ID NO:21.
An isolated nucleic acid molecule encoding a variant protein can be
created by introducing one or more nucleotide substitutions,
additions or deletions into the nucleotide sequence of SEQ ID NO:1,
2, 3, 13, 14, 19, 20, 31, 32, 39, 40, or 44 such that one or more
amino acid substitutions, additions or deletions are introduced
into the encoded protein. Mutations can be introduced by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Preferably, conservative amino acid substitutions are
made at one or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along all or
part of the coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for biological activity to
identify mutants that retain activity. Following mutagenesis, the
encoded protein can be expressed recombinantly and the activity of
the protein can be determined.
In a preferred embodiment, a mutant polypeptide that is a variant
of a polypeptide of the invention can be assayed for: (1) the
ability to form protein:protein interactions with proteins in a
signaling pathway of the polypeptide of the invention; (2) the
ability to bind a ligand of the polypeptide of the invention; or
(3) the ability to bind to an intracellular target protein of the
polypeptide of the invention. In yet another preferred embodiment,
the mutant polypeptide can be assayed for the ability to modulate
cellular proliferation, cellular migration or chemotaxis, or
cellular differentiation.
The present invention encompasses antisense nucleic acid molecules,
i.e., molecules which are complementary to a sense nucleic acid
encoding a polypeptide of the invention, e.g., complementary to the
coding strand of a double-stranded cDNA molecule or complementary
to an mRNA sequence. Accordingly, an antisense nucleic acid can
hydrogen bond to a sense nucleic acid. The antisense nucleic acid
can be complementary to an entire coding strand, or to only a
portion thereof, e.g., all or part of the protein coding region (or
open reading frame). An antisense nucleic acid molecule can be
antisense to all or part of a non-coding region of the coding
strand of a nucleotide sequence encoding a polypeptide of the
invention. The non-coding regions ("5' and 3' untranslated
regions") are the 5' and 3' sequences which flank the coding region
and are not translated into amino acids.
An antisense oligonucleotide can be, for example, about 5, 10, 15,
20, 25, 30, 35, 40, 45 or 50 nucleotides or more in length. An
antisense nucleic acid of the invention can be constructed using
chemical synthesis and enzymatic ligation reactions using
procedures known in the art. For example, an antisense nucleic acid
(e.g., an antisense oligonucleotide) can be chemically synthesized
using naturally occurring nucleotides or variously modified
nucleotides designed to increase the biological stability of the
molecules or to increase the physical stability of the duplex
formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate derivatives and acridine substituted nucleotides
can be used. Examples of modified nucleotides which can be used to
generate the antisense nucleic acid include 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a subject or generated in situ such that they
hybridize with or bind to cellular mRNA and/or genomic DNA encoding
a selected polypeptide of the invention to thereby inhibit
expression, e.g., by inhibiting transcription and/or translation.
The hybridization can be by conventional nucleotide complementarity
to form a stable duplex, or, for example, in the case of an
antisense nucleic acid molecule which binds to DNA duplexes,
through specific interactions in the major groove of the double
helix. An example of a route of administration of antisense nucleic
acid molecules of the invention includes direct injection at a
tissue site. Alternatively, antisense nucleic acid molecules can be
modified to target selected cells and then administered
systemically. For example, for systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface, e.g.,
by linking the antisense nucleic acid molecules to peptides or
antibodies which bind to cell surface receptors or antigens. The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong pol II or pol III promoter are
preferred.
An antisense nucleic acid molecule of the invention can be an
-anomeric nucleic acid molecule. An -anomeric nucleic acid molecule
forms specific double-stranded hybrids with complementary RNA in
which, contrary to the usual, .beta.-units, the strands run
parallel to each other (Gaultier et al. (1987) Nucleic Acids Res.
15:6625 6641). The antisense nucleic acid molecule can also
comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic
Acids Res. 15:6131 6148) or a chimeric RNA-DNA analogue (Inoue et
al. (1987) FEBS Lett. 215:327 330).
The invention also encompasses ribozymes. Ribozymes are catalytic
RNA molecules with ribonuclease activity which are capable of
cleaving a single-stranded nucleic acid, such as an mRNA, to which
they have a complementary region. Thus, ribozymes (e.g., hammerhead
ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585
591)) can be used to catalytically cleave mRNA transcripts to
thereby inhibit translation of the protein encoded by the mRNA. A
ribozyme having specificity for a nucleic acid molecule encoding a
polypeptide of the invention can be designed based upon the
nucleotide sequence of a cDNA disclosed herein. For example, a
derivative of a Tetrahymena L-19 IVS RNA can be constructed in
which the nucleotide sequence of the active site is complementary
to the nucleotide sequence to be cleaved in a Cech et al. U.S. Pat.
No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.
Alternatively, an mRNA encoding a polypeptide of the invention can
be used to select a catalytic RNA having a specific ribonuclease
activity from a pool of RNA molecules. See, e.g., Bartel and
Szostak (1993) Science 261:1411 1418.
The invention also encompasses nucleic acid molecules which form
triple helical structures. For example, expression of a polypeptide
of the invention can be inhibited by targeting nucleotide sequences
complementary to the regulatory region of the gene encoding the
polypeptide (e.g., the promoter and/or enhancer) to form triple
helical structures that prevent transcription of the gene in target
cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569
84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27 36; and Maher (1992)
Bioassays 14(12):807 15.
In various embodiments, the nucleic acid molecules of the invention
can be modified at the base moiety, sugar moiety or phosphate
backbone to improve, e.g., the stability, hybridization, or
solubility of the molecule. For example, the deoxyribose phosphate
backbone of the nucleic acids can be modified to generate peptide
nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4(1): 5 23). As used herein, the terms "peptide nucleic
acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in
which the deoxyribose phosphate backbone is replaced by a
pseudopeptide backbone and only the four natural nucleobases are
retained. The neutral backbone of PNAs has been shown to allow for
specific hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93: 14670 675.
PNAs can be used in therapeutic and diagnostic applications. For
example, PNAs can be used as antisense or antigene agents for
sequence-specific modulation of gene expression by, e.g., inducing
transcription or translation arrest or inhibiting replication. PNAs
can also be used, e.g., in the analysis of single base pair
mutations in a gene by, e.g., PNA directed PCR clamping; as
artificial restriction enzymes when used in combination with other
enzymes, e.g., S1 nucleases (Hyrup (1996), supra; or as probes or
primers for DNA sequence and hybridization (Hyrup (1996), supra;
Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670
675).
In another embodiment, PNAs can be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other
helper groups to PNA, by the formation of PNA-DNA chimeras, or by
the use of liposomes or other techniques of drug delivery known in
the art. For example, PNA-DNA chimeras can be generated which may
combine the advantageous properties of PNA and DNA. Such chimeras
allow DNA recognition enzymes, e.g., RNAse H and DNA polymerases,
to interact with the DNA portion while the PNA portion would
provide high binding affinity and specificity. PNA-DNA chimeras can
be linked using linkers of appropriate lengths selected in terms of
base stacking, number of bonds between the nucleobases, and
orientation (Hyrup (1996), supra). The synthesis of PNA-DNA
chimeras can be performed as described in Hyrup (1996), supra, and
Finn et al. (1996) Nucleic Acids Res. 24(17):3357 63. For example,
a DNA chain can be synthesized on a solid support using standard
phosphoramidite coupling chemistry and modified nucleoside analogs.
Compounds such as 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite can be used as a link between the PNA and the 5'
end of DNA (Mag et al. (1989) Nucleic Acids Res. 17:5973 88). PNA
monomers are then coupled in a stepwise manner to produce a
chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn
et al. (1996) Nucleic Acids Res. 24(17):3357 63). Alternatively,
chimeric molecules can be synthesized with a 5' DNA segment and a
3' PNA segment (Peterser et al. (1975) Bioorganic Med. Chem. Lett.
5:1119 11124).
In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al.(1989) Proc. Natl. Acad.
Sci. USA 86:6553 6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648 652; PCT Publication No. WO 88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
In addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (see, e.g., Krol et al.
(1988) Bio/Techniques 6:958 976) or intercalating agents (see,
e.g., Zon (1988) Pharm. Res. 5:539 549). To this end, the
oligonucleotide may be conjugated to another molecule, e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, hybridization-triggered cleavage agent, etc.
II. Isolated Proteins and Antibodies
One aspect of the invention pertains to isolated proteins, and
biologically active portions thereof, as well as polypeptide
fragments suitable for use as immunogens to raise antibodies
directed against a polypeptide of the invention. In one embodiment,
the native polypeptide can be isolated from cells or tissue sources
by an appropriate purification scheme using standard protein
purification techniques. In another embodiment, polypeptides of the
invention are produced by recombinant DNA techniques. Alternative
to recombinant expression, a polypeptide of the invention can be
synthesized chemically using standard peptide synthesis
techniques.
An "isolated" or "purified" protein or biologically active portion
thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein
(also referred to herein as a "contaminating protein"). When the
protein or biologically active portion thereof is recombinantly
produced, it is also preferably substantially free of culture
medium, i.e., culture medium represents less than about 20%, 10%,
or 5% of the volume of the protein preparation. When the protein is
produced by chemical synthesis, it is preferably substantially free
of chemical precursors or other chemicals, i.e., it is separated
from chemical precursors or other chemicals which are involved in
the synthesis of the protein. Accordingly such preparations of the
protein have less than about 30%, 20%, 10%, 5% (by dry weight) of
chemical precursors or compounds other than the polypeptide of
interest.
Biologically active portions of a polypeptide of the invention
include polypeptides comprising amino acid sequences sufficiently
identical to or derived from the amino acid sequence of the protein
(e.g., the amino acid sequence shown in any of SEQ ID NO:3, 4, 15,
16, 21, 22, 33, 34, 41, 42, or 45), which include fewer amino acids
than the full length protein, and exhibit at least one activity of
the corresponding full-length protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the corresponding protein. A biologically active
portion of a protein of the invention can be a polypeptide which
is, for example, 10, 25, 50, 100 or more amino acids in length.
Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of the native form of a polypeptide of the
invention.
Preferred polypeptides have the amino acid sequence of SEQ ID NO:3,
4, 15, 16, 21, 22, 33, 34, 41, 42, or 45. Other useful proteins are
substantially identical (e.g., at least about 45%, preferably 55%,
65%, 75%, 85%, 95%, or 99%) to any of SEQ ID NO: 3, 4, 15, 16, 21,
22, 33, 34, 41, 42, or 45, or substantially identical (e.g., at
least about 93%, preferably 94%, 95%, 96%, or 99%) to any of SEQ ID
NO:21 or 22, and retain the functional activity of the protein of
the corresponding naturally-occurring protein yet differ in amino
acid sequence due to natural allelic variation or mutagenesis.
To determine the percent identity of two amino acid sequences or of
two nucleic acids, the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in the sequence of a first
amino acid or nucleic acid sequence for optimal alignment with a
second amino or nucleic acid sequence). The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position. The percent identity between the
two sequences is a function of the number of identical positions
shared by the sequences (i.e., % identity=# of identical
positions/total # of positions (e.g., overlapping
positions).times.100). In one embodiment, the two sequences are the
same length.
The determination of percent identity between two sequences can be
accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264 2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873 5877.
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403 410.
BLAST nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to a nucleic acid molecules of the invention. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to a protein
molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25:3389 3402.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules (Id.). When
utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. See http://www.ncbi.nlm.nih.gov. Another preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller,
(1988) CABIOS 4:11 17. Such an algorithm is incorporated into the
ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used.
The percent identity between two sequences can be determined using
techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, only exact matches
are counted.
The invention also provides chimeric or fusion proteins. As used
herein, a "chimeric protein" or "fusion protein" comprises all or
part (preferably biologically active) of a polypeptide of the
invention operably linked to a heterologous polypeptide (i.e., a
polypeptide other than the same polypeptide of the invention).
Within the fusion protein, the term "operably linked" is intended
to indicate that the polypeptide of the invention and the
heterologous polypeptide are fused in-frame to each other. The
heterologous polypeptide can be fused to the N-terminus or
C-terminus of the polypeptide of the invention.
One useful fusion protein is a GST fusion protein in which the
polypeptide of the invention is fused to the C-terminus of GST
sequences. Such fusion proteins can facilitate the purification of
a recombinant polypeptide of the invention.
In another embodiment, the fusion protein contains a heterologous
signal sequence at its N-terminus. For example, the native signal
sequence of a polypeptide of the invention can be removed and
replaced with a signal sequence from another protein. For example,
the gp67 secretory sequence of the baculovirus envelope protein can
be used as a heterologous signal sequence (Current Protocols in
Molecular Biology, Ausubel et al., eds., John Wiley & Sons,
1992). Other examples of eukaryotic heterologous signal sequences
include the secretory sequences of melittin and human placental
alkaline phosphatase (Stratagene; La Jolla, Calif.). In yet another
example, useful prokaryotic heterologous signal sequences include
the phoA secretory signal (Sambrook et al., supra) and the protein
A secretory signal (Pharmacia Biotech; Piscataway, N.J.).
In yet another embodiment, the fusion protein is an immunoglobulin
fusion protein in which all or part of a polypeptide of the
invention is fused to sequences derived from a member of the
immunoglobulin protein family. The immunoglobulin fusion proteins
of the invention can be incorporated into pharmaceutical
compositions and administered to a subject to inhibit an
interaction between a ligand (soluble or membrane-bound) and a
protein on the surface of a cell (receptor), to thereby suppress
signal transduction in vivo. The immunoglobulin fusion protein can
be used to affect the bioavailability of a cognate ligand of a
polypeptide of the invention. Inhibition of ligand/receptor
interaction may be useful therapeutically, both for treating
proliferative and differentiative disorders and for modulating
(e.g., promoting or inhibiting) cell survival. Moreover, the
immunoglobulin fusion proteins of the invention can be used as
immunogens to produce antibodies directed against a polypeptide of
the invention in a subject, to purify ligands and in screening
assays to identify molecules which inhibit the interaction of
receptors with ligands.
Chimeric and fusion proteins of the invention can be produced by
standard recombinant DNA techniques. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, e.g., Ausubel et al.,
supra). Moreover, many expression vectors are commercially
available that already encode a fusion moiety (e.g., a GST
polypeptide). A nucleic acid encoding a polypeptide of the
invention can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the polypeptide of the
invention.
A signal sequence of a polypeptide of the invention (SEQ ID NO:5
and 19) can be used to facilitate secretion and isolation of the
secreted protein or other proteins of interest. Signal sequences
are typically characterized by a core of hydrophobic amino acids
which are generally cleaved from the mature protein during
secretion in one or more cleavage events. Such signal peptides
contain processing sites that allow cleavage of the signal sequence
from the mature proteins as they pass through the secretory
pathway. Thus, the invention pertains to the described polypeptides
having a signal sequence, as well as to the signal sequence itself
and to the polypeptide in the absence of the signal sequence (i.e.,
the cleavage products). In one embodiment, a nucleic acid sequence
encoding a signal sequence of the invention can be operably linked
in an expression vector to a protein of interest, such as a protein
which is ordinarily not secreted or is otherwise difficult to
isolate. The signal sequence directs secretion of the protein, such
as from a eukaryotic host into which the expression vector is
transformed, and the signal sequence is subsequently or
concurrently cleaved. The protein can then be readily purified from
the extracellular medium by art recognized methods. Alternatively,
the signal sequence can be linked to the protein of interest using
a sequence which facilitates purification, such as with a GST
domain.
In another embodiment, the signal sequences of the present
invention can be used to identify regulatory sequences, e.g.,
promoters, enhancers, repressors. Since signal sequences are the
most amino-terminal sequences of a peptide, it is expected that the
nucleic acids which flank the signal sequence on its amino-terminal
side will be regulatory sequences which affect transcription. Thus,
a nucleotide sequence which encodes all or a portion of a signal
sequence can be used as a probe to identify and isolate signal
sequences and their flanking regions, and these flanking regions
can be studied to identify regulatory elements therein.
The present invention also pertains to variants of the polypeptides
of the invention. Such variants have an altered amino acid sequence
which can function as either agonists (mimetics) or as antagonists.
Variants can be generated by mutagenesis, e.g., discrete point
mutation or truncation. An agonist can retain substantially the
same, or a subset, of the biological activities of the naturally
occurring form of the protein. An antagonist of a protein can
inhibit one or more of the activities of the naturally occurring
form of the protein by, for example, competitively binding to a
downstream or upstream member of a cellular signaling cascade which
includes the protein of interest. Thus, specific biological effects
can be elicited by treatment with a variant of limited function.
Treatment of a subject with a variant having a subset of the
biological activities of the naturally occurring form of the
protein can have fewer side effects in a subject relative to
treatment with the naturally occurring form of the protein.
Variants of a protein of the invention which function as either
agonists (mimetics) or as antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the protein of the invention for agonist or antagonist
activity. In one embodiment, a variegated library of variants is
generated by combinatorial mutagenesis at the nucleic acid level
and is encoded by a variegated gene library. A variegated library
of variants can be produced by, for example, enzymatically ligating
a mixture of synthetic oligonucleotides into gene sequences such
that a degenerate set of potential protein sequences is expressible
as individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display). There are a variety of
methods which can be used to produce libraries of potential
variants of the polypeptides of the invention from a degenerate
oligonucleotide sequence. Methods for synthesizing degenerate
oligonucleotides are known in the art (see, e.g., Narang (1983)
Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;
Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic
Acid Res. 11:477).
In addition, libraries of fragments of the coding sequence of a
polypeptide of the invention can be used to generate a variegated
population of polypeptides for screening and subsequent selection
of variants. For example, a library of coding sequence fragments
can be generated by treating a double stranded PCR fragment of the
coding sequence of interest with a nuclease under conditions
wherein nicking occurs only about once per molecule, denaturing the
double stranded DNA, renaturing the DNA to form double stranded DNA
which can include sense/antisense pairs from different nicked
products, removing single stranded portions from reformed duplexes
by treatment with S1 nuclease, and ligating the resulting fragment
library into an expression vector. By this method, an expression
library can be derived which encodes N-terminal and internal
fragments of various sizes of the protein of interest.
Several techniques are known in the art for screening gene products
of combinatorial libraries made by point mutations or truncation,
and for screening cDNA libraries for gene products having a
selected property. The most widely used techniques, which are
amenable to high through-put analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a technique
which enhances the frequency of functional mutants in the
libraries, can be used in combination with the screening assays to
identify variants of a protein of the invention (Arkin and Yourvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811 7815; Delgrave et al.
(1993) Protein Engineering 6(3):327 331).
An isolated polypeptide of the invention, or a fragment thereof,
can be used as an immunogen to generate antibodies using standard
techniques for polyclonal and monoclonal antibody preparation. The
full-length polypeptide or protein can be used or, alternatively,
the invention provides antigenic peptide fragments for use as
immunogens. The antigenic peptide of a protein of the invention
comprises at least 8 (preferably 10, 15, 20, or 30) amino acid
residues of the amino acid sequence of SEQ ID NO:3, 15, 21, or 45,
and encompasses an epitope of the protein such that an antibody
raised against the peptide forms a specific immune complex with the
protein.
Preferred epitopes encompassed by the antigenic peptide are regions
that are located on the surface of the protein, e.g., hydrophilic
regions. FIGS. 2, 7, and 10 are hydropathy plots of the proteins of
the invention. These plots or similar analyses can be used to
identify hydrophilic regions.
An immunogen typically is used to prepare antibodies by immunizing
a suitable subject, (e.g., rabbit, goat, mouse or other mammal). An
appropriate immunogenic preparation can contain, for example,
recombinantly expressed or chemically synthesized polypeptide. The
preparation can further include an adjuvant, such as Freund's
complete or incomplete adjuvant, or similar immunostimulatory
agent.
Accordingly, another aspect of the invention pertains to antibodies
directed against a polypeptide of the invention. The term
"antibody" as used herein refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site which specifically
binds an antigen, such as a polypeptide of the invention. A
molecule which specifically binds to a given polypeptide of the
invention is a molecule which binds the polypeptide, but does not
substantially bind other molecules in a sample, e.g., a biological
sample, which naturally contains the polypeptide. Examples of
immunologically active portions of immunoglobulin molecules include
F(ab) and F(ab').sub.2 fragments which can be generated by treating
the antibody with an enzyme such as pepsin. The invention provides
polyclonal and monoclonal antibodies. The term "monoclonal
antibody" or "monoclonal antibody composition", as used herein,
refers to a population of antibody molecules that contain only one
species of an antigen binding site capable of immunoreacting with a
particular epitope.
Polyclonal antibodies can be prepared as described above by
immunizing a suitable subject with a polypeptide of the invention
as an immunogen. The antibody titer in the immunized subject can be
monitored over time by standard techniques, such as with an enzyme
linked immunosorbent assay (ELISA) using immobilized polypeptide.
If desired, the antibody molecules can be isolated from the mammal
(e.g., from the blood) and further purified by well-known
techniques, such as protein A chromatography to obtain the IgG
fraction. At an appropriate time after immunization, e.g., when the
specific antibody titers are highest, antibody-producing cells can
be obtained from the subject and used to prepare monoclonal
antibodies by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein (1975) Nature 256:495
497, the human B cell hybridoma technique (Kozbor et al. (1983)
Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al.
(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77 96) or trioma techniques. The technology for producing
hybridomas is well known (see generally Current Protocols in
Immunology (1994) Coligan et al. (eds.) John Wiley & Sons,
Inc., New York, N.Y.). Hybridoma cells producing a monoclonal
antibody of the invention are detected by screening the hybridoma
culture supernatants for antibodies that bind the polypeptide of
interest, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas,
a monoclonal antibody directed against a polypeptide of the
invention can be identified and isolated by screening a recombinant
combinatorial immunoglobulin library (e.g., an antibody phage
display library) with the polypeptide of interest. Kits for
generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog No. 27-9400-01; and the Stratagene SurfZAP.TM. Phage
Display Kit, Catalog No. 240612). Additionally, examples of methods
and reagents particularly amenable for use in generating and
screening antibody display library can be found in, for example,
U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT
Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT
Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT
Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT
Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology
9:1370 1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81 85;
Huse et al. (1989) Science 246:1275 1281; Griffiths et al. (1993)
EMBO J. 12:725 734.
Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are within the scope of the invention. Such
chimeric and humanized monoclonal antibodies can be produced by
recombinant DNA techniques known in the art, for example using
methods described in PCT Publication No. WO 87/02671; European
Patent Application 184,187; European Patent Application 171,496;
European Patent Application 173,494; PCT Publication No. WO
86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et al. (1988) Science 240:1041 1043; Liu et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3439 3443; Liu et al. (1987)
J. Immunol. 139:3521 3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
USA 84:214 218; Nishimura et al. (1987) Canc. Res. 47:999 1005;
Wood et al. (1985) Nature 314:446 449; and Shaw et al. (1988) J.
Natl. Cancer Inst. 80:1553 1559); Morrison (1985) Science 229:1202
1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No.
5,225,539; Jones et al. (1986) Nature 321:552 525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053 4060.
Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Such antibodies can be
produced using transgenic mice which are incapable of expressing
endogenous immunoglobulin heavy and light chains genes, but which
can express human heavy and light chain genes. The transgenic mice
are immunized in the normal fashion with a selected antigen, e.g.,
all or a portion of a polypeptide of the invention. Monoclonal
antibodies directed against the antigen can be obtained using
conventional hybridoma technology. The human immunoglobulin
transgenes harbored by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and
somatic mutation. Thus, using such a technique, it is possible to
produce therapeutically useful IgG, IgA and IgE antibodies. For an
overview of this technology for producing human antibodies, see
Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65 93). For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S.
Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No.
5,661,016; and U.S. Pat. No. 5,545,806. In addition, companies such
as Abgenix, Inc. (Freemont, Calif.), can be engaged to provide
human antibodies directed against a selected antigen using
technology similar to that described above.
Completely human antibodies which recognize a selected epitope can
be generated using a technique referred to as "guided selection."
In this approach a selected non-human monoclonal antibody, e.g., a
murine antibody, is used to guide the selection of a completely
human antibody recognizing the same epitope. (Jespers et al. (1994)
Bio/technology 12:899 903).
An antibody directed against a polypeptide of the invention (e.g.,
monoclonal antibody) can be used to isolate the polypeptide by
standard techniques, such as affinity chromatography or
immunoprecipitation. Moreover, such an antibody can be used to
detect the protein (e.g., in a cellular lysate or cell supernatant)
in order to evaluate the abundance and pattern of expression of the
polypeptide. The antibodies can also be used diagnostically to
monitor protein levels in tissue as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, beta-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
III. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably
expression vectors, containing a nucleic acid encoding a
polypeptide of the invention (or a portion thereof). As used
herein, the term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
One type of vector is a "plasmid", which refers to a circular
double stranded DNA loop into which additional DNA segments can be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments can be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) are integrated
into the genome of a host cell upon introduction into the host
cell, and thereby are replicated along with the host genome.
Moreover, certain vectors, expression vectors, are capable of
directing the expression of genes to which they are operably
linked. In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids (vectors).
However, the invention is intended to include such other forms of
expression vectors, such as viral vectors (e.g., replication
defective retroviruses, adenoviruses and adeno-associated viruses),
which serve equivalent functions.
The recombinant expression vectors of the invention comprise a
nucleic acid of the invention in a form suitable for expression of
the nucleic acid in a host cell. This means that the recombinant
expression vectors include one or more regulatory sequences,
selected on the basis of the host cells to be used for expression,
which is operably linked to the nucleic acid sequence to be
expressed. Within a recombinant expression vector, "operably
linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory sequence(s) in a manner which
allows for expression of the nucleotide sequence (e.g., in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, etc. The
expression vectors of the invention can be introduced into host
cells to thereby produce proteins or peptides, including fusion
proteins or peptides, encoded by nucleic acids as described
herein.
The recombinant expression vectors of the invention can be designed
for expression of a polypeptide of the invention in prokaryotic
(e.g., E. coli) or eukaryotic cells (e.g., insect cells (using
baculovirus expression vectors), yeast cells or mammalian cells).
Suitable host cells are discussed further in Goeddel, supra.
Alternatively, the recombinant expression vector can be transcribed
and translated in vitro, for example using T7 promoter regulatory
sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in
E. coli with vectors containing constitutive or inducible promoters
directing the expression of either fusion or non-fusion proteins.
Fusion vectors add a number of amino acids to a protein encoded
therein, usually to the amino terminus of the recombinant protein.
Such fusion vectors typically serve three purposes: 1) to increase
expression of recombinant protein; 2) to increase the solubility of
the recombinant protein; and 3) to aid in the purification of the
recombinant protein by acting as a ligand in affinity purification.
Often, in fusion expression vectors, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson
(1988) Gene 67:31 40), pMAL (New England Biolabs, Beverly, Mass.)
and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione
S-transferase (GST), maltose E binding protein, or protein A,
respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301 315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60 89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
.lamda. prophage harboring a T7 gn1 gene under the transcriptional
control of the lacUV 5 promoter.
One strategy to maximize recombinant protein expression in E. coli
is to express the protein in a host bacteria with an impaired
capacity to proteolytically cleave the recombinant protein
(Gottesman, Gene Expression Technology: Methods in Enzymology 185,
Academic Press San Diego, Calif. (1990) 119 428). Another strategy
is to alter the nucleic acid sequence of the nucleic acid to be
inserted into an expression vector so that the individual codons
for each amino acid are those preferentially utilized in E. coli
(Wada et al. (1992) Nucleic Acids Res. 20:2111 2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
In another embodiment, the expression vector is a yeast expression
vector. Examples of vectors for expression in yeast S. cerivisae
include pYepSec1 (Baldari et al. (1987) EMBO J. 6:229 234), pMFa
(Kurjan and Herskowitz, (1982) Cell 30:933 943), pJRY88 (Schultz et
al. (1987) Gene 54:113 123), pYES2 (Invitrogen Corporation, San
Diego, Calif.), and pPicZ (Invitrogen Corp, San Diego, Calif.).
Alternatively, the expression vector is a baculovirus expression
vector. Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al. (1983) Mol. Cell Biol. 3:2156 2165) and the pVL
series (Lucklow and Summers (1989) Virology 170:31 39).
In yet another embodiment, a nucleic acid of the invention is
expressed in mammalian cells using a mammalian expression vector.
Examples of mammalian expression vectors include pCDM8 (Seed (1987)
Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187
195). When used in mammalian cells, the expression vector's control
functions are often provided by viral regulatory elements. For
example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40. For other
suitable expression systems for both prokaryotic and eukaryotic
cells see chapters 16 and 17 of Sambrook et al., supra.
In another embodiment, the recombinant mammalian expression vector
is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268 277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235 275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729 733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729 740; Queen and
Baltimore (1983) Cell 33:741 748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473 5477), pancreas-specific promoters (Edlund
et al. (1985) Science 230:912 916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss (1990) Science
249:374 379) and the beta-fetoprotein promoter (Campes and Tilghman
(1989) Genes Dev. 3:537 546).
The invention further provides a recombinant expression vector
comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operably linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to the mRNA encoding a
polypeptide of the invention. Regulatory sequences operably linked
to a nucleic acid cloned in the antisense orientation can be chosen
which direct the continuous expression of the antisense RNA
molecule in a variety of cell types, for instance viral promoters
and/or enhancers, or regulatory sequences can be chosen which
direct constitutive, tissue specific or cell type specific
expression of antisense RNA. The antisense expression vector can be
in the form of a recombinant plasmid, phagemid or attenuated virus
in which antisense nucleic acids are produced under the control of
a high efficiency regulatory region, the activity of which can be
determined by the cell type into which the vector is introduced.
For a discussion of the regulation of gene expression using
antisense genes see Weintraub et al. (Reviews--Trends in Genetics,
Vol. 1(1) 1986).
Another aspect of the invention pertains to host cells into which a
recombinant expression vector of the invention has been introduced.
The terms "host cell" and "recombinant host cell" are used
interchangeably herein. It is understood that such terms refer not
only to the particular subject cell but to the progeny or potential
progeny of such a cell. Because certain modifications may occur in
succeeding generations due to either mutation or
environmental-influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic
cell (e.g., insect cells, yeast or mammalian cells).
Vector DNA can be introduced into prokaryotic or eukaryotic cells
via conventional transformation or transfection techniques. As used
herein, the terms "transformation" and "transfection" are intended
to refer to a variety of art-recognized techniques for introducing
foreign nucleic acid into a host cell, including calcium phosphate
or calcium chloride co-precipitation, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Suitable methods for
transforming or transfecting host cells can be found in Sambrook,
et al. (supra), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that,
depending upon the expression vector and transfection technique
used, only a small fraction of cells may integrate the foreign DNA
into their genome. In order to identify and select these
integrants, a gene that encodes a selectable marker (e.g., for
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Cells stably transfected with the
introduced nucleic acid can be identified by drug selection (e.g.,
cells that have incorporated the selectable marker gene will
survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic
host cell in culture, can be used to produce a polypeptide of the
invention. Accordingly, the invention further provides methods for
producing a polypeptide of the invention using the host cells of
the invention. In one embodiment, the method comprises culturing
the host cell of invention (into which a recombinant expression
vector encoding a polypeptide of the invention has been introduced)
in a suitable medium such that the polypeptide is produced. In
another embodiment, the method further comprises isolating the
polypeptide from the medium or the host cell.
The host cells of the invention can also be used to produce
nonhuman transgenic animals. For example, in one embodiment, a host
cell of the invention is a fertilized oocyte or an embryonic stem
cell into which a sequence encoding a polypeptide of the invention
has been introduced. Such host cells can then be used to create
non-human transgenic animals in which exogenous sequences encoding
a polypeptide of the invention have been introduced into their
genome or homologous recombinant animals in which endogenous
encoding a polypeptide of the invention sequences have been
altered. Such animals are useful for studying the function and/or
activity of the polypeptide and for identifying and/or evaluating
modulators of polypeptide activity. As used herein, a "transgenic
animal" is a non-human animal, preferably a mammal, more preferably
a rodent such as a rat or mouse, in which one or more of the cells
of the animal includes a transgene. Other examples of transgenic
animals include non-human primates, sheep, dogs, cows, goats,
chickens, amphibians, etc. A transgene is exogenous DNA which is
integrated into the genome of a cell from which a transgenic animal
develops and which remains in the genome of the mature animal,
thereby directing the expression of an encoded gene product in one
or more cell types or tissues of the transgenic animal. As used
herein, an "homologous recombinant animal" is a non-human animal,
preferably a mammal, more preferably a mouse, in which an
endogenous gene has been altered by homologous recombination
between the endogenous gene and an exogenous DNA molecule
introduced into a cell of the animal, e.g., an embryonic cell of
the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing
nucleic acid encoding a polypeptide of the invention (or a
homologue thereof) into the male pronuclei of a fertilized oocyte,
e.g., by microinjection, retroviral infection, and allowing the
oocyte to develop in a pseudopregnant female foster animal.
Intronic sequences and polyadenylation signals can also be included
in the transgene to increase the efficiency of expression of the
transgene. A tissue-specific regulatory sequence(s) can be operably
linked to the transgene to direct expression of the polypeptide of
the invention to particular cells. Methods for generating
transgenic animals via embryo manipulation and microinjection,
particularly animals such as mice, have become conventional in the
art and are described, for example, in U.S. Pat. Nos. 4,736,866 and
4,870,009, U.S. Pat. No. 4,873,191 and in Hogan, Manipulating the
Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1986). Similar methods are used for production of
other transgenic animals. A transgenic founder animal can be
identified based upon the presence of the transgene in its genome
and/or expression of mRNA encoding the transgene in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying the transgene can further be bred to
other transgenic animals carrying other transgenes.
To create an homologous recombinant animal, a vector is prepared
which contains at least a portion of a gene encoding a polypeptide
of the invention into which a deletion, addition or substitution
has been introduced to thereby alter, e.g., functionally disrupt,
the gene. In a preferred embodiment, the vector is designed such
that, upon homologous recombination, the endogenous gene is
functionally disrupted (i.e., no longer encodes a functional
protein; also referred to as a "knock out" vector). Alternatively,
the vector can be designed such that, upon homologous
recombination, the endogenous gene is mutated or otherwise altered
but still encodes functional protein (e.g., the upstream regulatory
region can be altered to thereby alter the expression of the
endogenous protein). In the homologous recombination vector, the
altered portion of the gene is flanked at its 5' and 3' ends by
additional nucleic acid of the gene to allow for homologous
recombination to occur between the exogenous gene carried by the
vector and an endogenous gene in an embryonic stem cell. The
additional flanking nucleic acid sequences are of sufficient length
for successful homologous recombination with the endogenous gene.
Typically, several kilobases of flanking DNA (both at the 5' and 3'
ends) are included in the vector (see, e.g., Thomas and Capecchi
(1987) Cell 51:503 for a description of homologous recombination
vectors). The vector is introduced into an embryonic stem cell line
(e.g., by electroporation) and cells in which the introduced gene
has homologously recombined with the endogenous gene are selected
(see, e.g., Li et al. (1992) Cell 69:915). The selected cells are
then injected into a blastocyst of an animal (e.g., a mouse) to
form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas
and Embryonic Stem Cells: A Practical Approach, Robertson, ed.
(IRL, Oxford, 1987) pp. 113 152). A chimeric embryo can then be
implanted into a suitable pseudopregnant female foster animal and
the embryo brought to term. Progeny harboring the homologously
recombined DNA in their germ cells can be used to breed animals in
which all cells of the animal contain the homologously recombined
DNA by germline transmission of the transgene. Methods for
constructing homologous recombination vectors and homologous
recombinant animals are described further in Bradley (1991) Current
Opinion in Bio/Technology 2:823 829 and in PCT Publication Nos. WO
90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.
In another embodiment, transgenic non-human animals can be produced
which contain selected systems which allow for regulated expression
of the transgene. One example of such a system is the cre/loxP
recombinase system of bacteriophage P1. For a description of the
cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc.
Natl. Acad. Sci. USA 89:6232 6236. Another example of a recombinase
system is the FLP recombinase system of Saccharomyces cerevisiae
(O'Gorman et al. (1991) Science 251:1351 1355. If a cre/loxP
recombinase system is used to regulate expression of the transgene,
animals containing transgenes encoding both the Cre recombinase and
a selected protein are required. Such animals can be provided
through the construction of "double" transgenic animals, e.g., by
mating two transgenic animals, one containing a transgene encoding
a selected protein and the other containing a transgene encoding a
recombinase.
Clones of the non-human transgenic animals described herein can
also be produced according to the methods described in Wilmut et
al. (1997) Nature 385:810 813 and PCT Publication NOS. WO 97/07668
and WO 97/07669.
IV. Pharmaceutical Compositions
The nucleic acid molecules, polypeptides, and antibodies (also
referred to herein as "active compounds") of the invention can be
incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise the nucleic
acid molecule, protein, or antibody and a pharmaceutically
acceptable carrier. As used herein the language "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
The invention includes methods for preparing pharmaceutical
compositions for modulating the expression or activity of a
polypeptide or nucleic acid of the invention. Such methods comprise
formulating a pharmaceutically acceptable carrier with an agent
which modulates expression or activity of a polypeptide or nucleic
acid of the invention. Such compositions can further include
additional active agents. Thus, the invention further includes
methods for preparing a pharmaceutical composition by formulating a
pharmaceutically acceptable carrier with an agent which modulates
expression or activity of a polypeptide or nucleic acid of the
invention and one or more additional active compounds.
A pharmaceutical composition of the invention is formulated to be
compatible with its intended route of administration. Examples of
routes of administration include parenteral, e.g., intravenous,
intradermal, subcutaneous, oral (e.g., inhalation), transdermal
(topical), transmucosal, and rectal administration. Solutions or
suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
Pharmaceutical compositions suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF; Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
Sterile injectable solutions can be prepared by incorporating the
active compound (e.g., a polypeptide or antibody) in the required
amount in an appropriate solvent with one or a combination of
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the-preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible
carrier. They can be enclosed in gelatin capsules or compressed
into tablets. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules. Oral compositions can
also be prepared using a fluid carrier for use as a mouthwash,
wherein the compound in the fluid carrier is applied orally and
swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
For administration by inhalation, the compounds are delivered in
the form of an aerosol spray from a pressurized container or
dispenser which contains a suitable propellant, e.g., a gas such as
carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal
means. For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art, and
include, for example, for transmucosal administration, detergents,
bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
The compounds can also be prepared in the form of suppositories
(e.g., with conventional suppository bases such as cocoa butter and
other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers
that will protect the compound against rapid elimination from the
body, such as a controlled release formulation, including implants
and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
It is especially advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on the unique characteristics of
the active compound and the particular therapeutic effect to be
achieved, and the limitations inherent in the art of compounding
such an active compound for the treatment of individuals.
For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of
body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to
act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually
appropriate. Generally, partially human antibodies and fully human
antibodies have a longer half-life within the human body than other
antibodies. Accordingly, lower dosages and less frequent
administration is often possible. Modifications such as lipidation
can be used to stabilize antibodies and to enhance uptake and
tissue penetration (e.g., into the brain). A method for lipidation
of antibodies is described by Cruikshank et al. ((1997) J. Acquired
Immune Deficiency Syndromes and Human Retrovirology 14:193).
The nucleic acid molecules of the invention can be inserted into
vectors and used as gene therapy vectors. Gene therapy vectors can
be delivered to a subject by, for example, intravenous injection,
local administration (U.S. Pat. No. 5,328,470) or by stereotactic
injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA
91:3054 3057). The pharmaceutical preparation of the gene therapy
vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. Alternatively, where the complete
gene delivery vector can be produced intact from recombinant cells,
e.g., retroviral vectors, the pharmaceutical preparation can
include one or more cells which produce the gene delivery
system.
The pharmaceutical compositions can be included in a container,
pack, or dispenser together with instructions for
administration.
V. Uses and Methods of the Invention
The nucleic acid molecules, proteins, protein homologues, and
antibodies described herein can be used in one or more of the
following methods: a) screening assays; b) detection assays (e.g.,
chromosomal mapping, tissue typing, forensic biology); c)
predictive medicine (e.g., diagnostic assays, prognostic assays,
monitoring clinical trials, and pharmacogenomics); and d) methods
of treatment (e.g., therapeutic and prophylactic). The isolated
nucleic acid molecules of the invention can be used to express
proteins (e.g., via a recombinant expression vector in a host cell
in gene therapy applications), to detect mRNA (e.g., in a
biological sample) or a genetic lesion, and to modulate activity of
a polypeptide of the invention. In addition, the polypeptides of
the invention can be used to screen drugs or compounds which
modulate activity or expression of a polypeptide of the invention
as well as to treat disorders characterized by insufficient or
excessive production of a protein of the invention or production of
a form of a protein of the invention which has decreased or
aberrant activity compared to the wild type protein. In addition,
the antibodies of the invention can be used to detect and isolate a
protein of the and modulate activity of a protein of the
invention.
This invention further pertains to novel agents identified by the
above-described screening assays and uses thereof for treatments as
described herein.
A. Screening Assays
The invention provides a method (also referred to herein as a
"screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules or other drugs) which bind to polypeptide of the
invention or have a stimulatory or inhibitory effect on, for
example, expression or activity of a polypeptide of the
invention.
In one embodiment, the invention provides assays for screening
candidate or test compounds which bind to or modulate the activity
of the membrane-bound form of a polypeptide of the invention or
biologically active portion thereof. The test compounds of the
present invention can be obtained using any of the numerous
approaches in combinatorial library methods known in the art,
including: biological libraries; spatially addressable parallel
solid phase or solution phase libraries; synthetic library methods
requiring deconvolution; the "one-bead one-compound" library
method; and synthetic library methods using affinity chromatography
selection. The biological library approach is limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam (1997) Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be
found in the art, for example in: DeWitt et al. (1993) Proc. Natl.
Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci.
USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho
et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed.
Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.
Libraries of compounds may be presented in solution (e.g., Houghten
(1992) Bio/Techniques 13:412 421), or on beads (Lam (1991) Nature
354:82 84), chips (Fodor (1993) Nature 364:555 556), bacteria (U.S.
Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484;
and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci.
USA 89:1865 1869) or phage (Scott and Smith (1990) Science 249:386
390; Devlin (1990) Science 249:404 406; Cwirla et al. (1990) Proc.
Natl. Acad. Sci. USA 87:6378 6382; and Felici (1991) J. Mol. Biol.
222:301 310).
In one embodiment, an assay is a cell-based assay in which a cell
which expresses a membrane-bound form of a polypeptide of the
invention, or a biologically active portion thereof, on the cell
surface is contacted with a test compound and the ability of the
test compound to bind to the polypeptide determined. The cell, for
example, can be a yeast cell or a cell of mammalian origin.
Determining the ability of the test compound to bind to the
polypeptide can be accomplished, for example, by coupling the test
compound with a radioisotope or enzymatic label such that binding
of the test compound to the polypeptide or biologically active
portion thereof can be determined by detecting the labeled compound
in a complex. For example, test compounds can be labeled with
.sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Alternatively, test
compounds can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic label detected by determination of conversion of an
appropriate substrate to product. In a preferred embodiment, the
assay comprises contacting a cell which expresses a membrane-bound
form of a polypeptide of the invention, or a biologically active
portion thereof, on the cell surface with a known compound which
binds the polypeptide to form an assay mixture, contacting the
assay mixture with a test compound, and determining the ability of
the test compound to interact with the polypeptide, wherein
determining the ability of the test compound to interact with the
polypeptide comprises determining the ability of the test compound
to preferentially bind to the polypeptide or a biologically active
portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising
contacting a cell expressing a membrane-bound form of a polypeptide
of the invention, or a biologically active portion thereof, on the
cell surface with a test compound and determining the ability of
the test compound to modulate (e.g., stimulate or inhibit) the
activity of the polypeptide or biologically active portion thereof.
Determining the ability of the test compound to modulate the
activity of the polypeptide or a biologically active portion
thereof can be accomplished, for example, by determining the
ability of the polypeptide protein to bind to or interact with a
target molecule.
Determining the ability of a polypeptide of the invention to bind
to or interact with a target molecule can be accomplished by one of
the methods described above for determining direct binding. As used
herein, a "target molecule" is a molecule with which a selected
polypeptide (e.g., a polypeptide of the invention) binds or
interacts with in nature, for example, a molecule on the surface of
a cell which expresses the selected protein, a molecule on the
surface of a second cell, a molecule in the extracellular milieu, a
molecule associated with the internal surface of a cell membrane or
a cytoplasmic molecule. A target molecule can be a polypeptide of
the invention or some other polypeptide or protein. For example, a
target molecule can be a component of a signal transduction pathway
which facilitates transduction of an extracellular signal (e.g., a
signal generated by binding of a compound to a polypeptide of the
invention) through the cell membrane and into the cell or a second
intercellular protein which has catalytic activity or a protein
which facilitates the association of downstream signaling molecules
with a polypeptide of the invention. Determining the ability of a
polypeptide of the invention to bind to or interact with a target
molecule can be accomplished by determining the activity of the
target molecule. For example, the activity of the target molecule
can be determined by detecting induction of a cellular second
messenger of the target (e.g., intracellular Ca.sup.2+,
diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity
of the target on an appropriate substrate, detecting the induction
of a reporter gene (e.g., a regulatory element that is responsive
to a polypeptide of the invention operably linked to a nucleic acid
encoding a detectable marker, e.g., luciferase), or detecting a
cellular response, for example, cellular differentiation, or cell
proliferation.
In yet another embodiment, an assay of the present invention is a
cell-free assay comprising contacting a polypeptide of the
invention or biologically active portion thereof with a test
compound and determining the ability of the test compound to bind
to the polypeptide or biologically active portion thereof. Binding
of the test compound to the polypeptide can be determined either
directly or indirectly as described above. In a preferred
embodiment, the assay includes contacting the polypeptide of the
invention or biologically active portion thereof with a known
compound which binds the polypeptide to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with the polypeptide,
wherein determining the ability of the test compound to interact
with the polypeptide comprises determining the ability of the test
compound to preferentially bind to the polypeptide or biologically
active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-free assay comprising
contacting a polypeptide of the invention or biologically active
portion thereof with a test compound and determining the ability of
the test compound to modulate (e.g., stimulate or inhibit) the
activity of the polypeptide or biologically active portion thereof.
Determining the ability of the test compound to modulate the
activity of the polypeptide can be accomplished, for example, by
determining the ability of the polypeptide to bind to a target
molecule by one of the methods described above for determining
direct binding. In an alternative embodiment, determining the
ability of the test compound to modulate the activity of the
polypeptide can be accomplished by determining the ability of the
polypeptide of the invention to further modulate the target
molecule. For example, the catalytic/enzymatic activity of the
target molecule on an appropriate substrate can be determined as
previously described.
In yet another embodiment, the cell-free assay comprises contacting
a polypeptide of the invention or biologically active portion
thereof with a known compound which binds the polypeptide to form
an assay mixture, contacting the assay mixture with a test
compound, and determining the ability of the test compound to
interact with the polypeptide, wherein determining the ability of
the test compound to interact with the polypeptide comprises
determining the ability of the polypeptide to preferentially bind
to or modulate the activity of a target molecule.
The cell-free assays of the present invention are amenable to use
of both a soluble form or the membrane-bound form of a polypeptide
of the invention. In the case of cell-free assays comprising the
membrane-bound form of the polypeptide, it may be desirable to
utilize a solubilizing agent such that the membrane-bound form of
the polypeptide is maintained in solution. Examples of such
solubilizing agents include non-ionic detergents such as
n-octylglucoside, n-dodecylglucoside, n-octylmaltoside,
octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton
X-100, Triton X-114, Thesit, Isotridecypoly(ethylene glycol
ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate
(CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or
N-dodecyl.dbd.N,N-dimethyl-3-ammonio-1-propane sulfonate.
In more than one embodiment of the above assay methods of the
present invention, it may be desirable to immobilize either the
polypeptide of the invention or its target molecule to facilitate
separation of complexed from uncomplexed forms of one or both of
the proteins, as well as to accommodate automation of the assay.
Binding of a test compound to the polypeptide, or interaction of
the polypeptide with a target molecule in the presence and absence
of a candidate compound, can be accomplished in any vessel suitable
for containing the reactants. Examples of such vessels include
microtitre plates, test tubes, and micro-centrifuge tubes. In one
embodiment, a fusion protein can be provided which adds a domain
that allows one or both of the proteins to be bound to a matrix.
For example, glutathione-S-transferase fusion proteins or
glutathione-S-transferase fusion proteins can be adsorbed onto
glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or
glutathione derivatized microtitre plates, which are then combined
with the test compound or the test compound and either the
non-adsorbed target protein or A polypeptide of the invention, and
the mixture incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and pH).
Following incubation, the beads or microtitre plate wells are
washed to remove any unbound components and complex formation is
measured either directly or indirectly, for example, as described
above. Alternatively, the complexes can be dissociated from the
matrix, and the level of binding or activity of the polypeptide of
the invention can be determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be
used in the screening assays of the invention. For example, either
the polypeptide of the invention or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated polypeptide of the invention or target molecules can
be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques
well known in the art (e.g., biotinylation kit, Pierce Chemicals;
Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies reactive with the polypeptide of the
invention or target molecules but which do not interfere with
binding of the polypeptide of the invention to its target molecule
can be derivatized to the wells of the plate, and unbound target or
polypeptide of the invention trapped in the wells by antibody
conjugation. Methods for detecting such complexes, in addition to
those described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the
polypeptide of the invention or target molecule, as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the polypeptide of the invention or target
molecule.
In another embodiment, modulators of expression of a polypeptide of
the invention are identified in a method in which a cell is
contacted with a candidate compound and the expression of the
selected mRNA or protein (i.e., the mRNA or protein corresponding
to a polypeptide or nucleic acid of the invention) in the cell is
determined. The level of expression of the selected mRNA or protein
in the presence of the candidate compound is compared to the level
of expression of the selected mRNA or protein in the absence of the
candidate compound. The candidate compound can then be identified
as a modulator of expression of the polypeptide of the invention
based on this comparison. For example, when expression of the
selected mRNA or protein is greater (statistically significantly
greater) in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of
the selected mRNA or protein expression. Alternatively, when
expression of the selected mRNA or protein is less (statistically
significantly less) in the presence of the candidate compound than
in its absence, the candidate compound is identified as an
inhibitor of the selected mRNA or protein expression. The level of
the selected mRNA or protein expression in the cells can be
determined by methods described herein.
In yet another aspect of the invention, a polypeptide of the
inventions can be used as "bait proteins" in a two-hybrid assay or
three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223 232; Madura et al. (1993) J. Biol. Chem.
268:12046 12054; Bartel et al. (1993) Bio/Techniques 14:920 924;
Iwabuchi et al. (1993) Oncogene 8:1693 1696; and PCT Publication
No. WO 94/10300), to identify other proteins, which bind to or
interact with the polypeptide of the invention and modulate
activity of the polypeptide of the invention. Such binding proteins
are also likely to be involved in the propagation of signals by the
polypeptide of the inventions as, for example, upstream or
downstream elements of a signaling pathway involving the
polypeptide of the invention.
This invention further pertains to novel agents identified by the
above-described screening assays and uses thereof for treatments as
described herein.
B. Detection Assays
Portions or fragments of the cDNA sequences identified herein (and
the corresponding complete gene sequences) can be used in numerous
ways as polynucleotide reagents. For example, these sequences can
be used to: (i) map their respective genes on a chromosome and,
thus, locate gene regions associated with genetic disease; (ii)
identify an individual from a minute biological sample (tissue
typing); and (iii) aid in forensic identification of a biological
sample. These applications are described in the subsections
below.
1. Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been
isolated, this sequence can be used to map the location of the gene
on a chromosome. Accordingly, nucleic acid molecules described
herein or fragments thereof, can be used to map the location of the
corresponding genes on a chromosome. The mapping of the sequences
to chromosomes is an important first step in correlating these
sequences with genes associated with disease.
Briefly, genes can be mapped to chromosomes by preparing PCR
primers (preferably 15 25 bp in length) from the sequence of a gene
of the invention. Computer analysis of the sequence of a gene of
the invention can be used to rapidly select primers that do not
span more than one exon in the genomic DNA, thus complicating the
amplification process. These primers can then be used for PCR
screening of somatic cell hybrids containing individual human
chromosomes. Only those hybrids containing the human gene
corresponding to the gene sequences will yield an amplified
fragment. For a review of this technique, see D'Eustachio et al.
((1983) Science 220:919 924).
PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the nucleic acid sequences of the invention to design
oligonucleotide primers, sublocalization can be achieved with
panels of fragments from specific chromosomes. Other mapping
strategies which can similarly be used to map a gene to its
chromosome include in situ hybridization (described in Fan et al.
(1990) Proc. Natl. Acad. Sci. USA 87:6223 27), pre-screening with
labeled flow-sorted chromosomes (CITE), and pre-selection by
hybridization to chromosome specific cDNA libraries. Fluorescence
in situ hybridization (FISH) of a DNA sequence to a metaphase
chromosomal spread can further be used to provide a precise
chromosomal location in one step. For a review of this technique,
see Verma et al., (Human Chromosomes: A Manual of Basic Techniques
(Pergamon Press, New York, 1988)).
Reagents for chromosome mapping can be used individually to mark a
single chromosome or a single site on that chromosome, or panels of
reagents can be used for marking multiple sites and/or multiple
chromosomes. Reagents corresponding to noncoding regions of the
genes actually are preferred for mapping purposes. Coding sequences
are more likely to be conserved within gene families, thus
increasing the chance of cross hybridizations during chromosomal
mapping.
Once a sequence has been mapped to a precise chromosomal location,
the physical position of the sequence on the chromosome can be
correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between genes and disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, e.g.,
Egeland et al. (1987) Nature 325:783 787.
Moreover, differences in the DNA sequences between individuals
affected and unaffected with a disease associated with a gene of
the invention can be determined. If a mutation is observed in some
or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
2. Tissue Typing
The nucleic acid sequences of the present invention can also be
used to identify individuals from minute biological samples. The
United States military, for example, is considering the use of
restriction fragment length polymorphism (RFLP) for identification
of its personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identification. This method
does not suffer from the current limitations of "Dog Tags" which
can be lost, switched, or stolen, making positive identification
difficult. The sequences of the present invention are useful as
additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
Furthermore, the sequences of the present invention can be used to
provide an alternative technique which determines the actual
base-by-base DNA sequence of selected portions of an individual's
genome. Thus, the nucleic acid sequences described herein can be
used to prepare two PCR primers from the 5' and 3' ends of the
sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in
this manner, can provide unique individual identifications, as each
individual will have a unique set of such DNA sequences due to
allelic differences. The sequences of the present invention can be
used to obtain such identification sequences from individuals and
from tissue. The nucleic acid sequences of the invention uniquely
represent portions of the human genome. Allelic variation occurs to
some degree in the coding regions of these sequences, and to a
greater degree in the noncoding regions. It is estimated that
allelic variation between individual humans occurs with a frequency
at about once per each 500 bases. Each of the sequences described
herein can, to some degree, be used as a standard against which DNA
from an individual can be compared for identification purposes.
Because greater numbers of polymorphisms occur in the noncoding
regions, fewer sequences are necessary to differentiate
individuals. The noncoding sequences of SEQ ID NO:1, 13, or 19 can
comfortably provide positive individual identification with a panel
of perhaps 10 to 1,000 primers which each yield a noncoding
amplified sequence of 100 bases. If predicted coding sequences,
such as those in SEQ ID NO:2, 14, 20, or 44 are used, a more
appropriate number of primers for positive individual
identification would be 500 2,000.
If a panel of reagents from the nucleic acid sequences described
herein is used to generate a unique identification database for an
individual, those same reagents can later be used to identify
tissue from that individual. Using the unique identification
database, positive identification of the individual, living or
dead, can be made from extremely small tissue samples.
3. Use of Partial Gene Sequences in Forensic Biology
DNA-based identification techniques can also be used in forensic
biology. Forensic biology is a scientific field employing genetic
typing of biological evidence found at a crime scene as a means for
positively identifying, for example, a perpetrator of a crime. To
make such an identification, PCR technology can be used to amplify
DNA sequences taken from very small biological samples such as
tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva,
or semen found at a crime scene. The amplified sequence can then be
compared to a standard, thereby allowing identification of the
origin of the biological sample.
The sequences of the present invention can be used to provide
polynucleotide reagents, e.g., PCR primers, targeted to specific
loci in the human genome, which can enhance the reliability of
DNA-based forensic identifications by, for example, providing
another "identification marker" (i.e. another DNA sequence that is
unique to a particular individual). As mentioned above, actual base
sequence information can be used for identification as an accurate
alternative to patterns formed by restriction enzyme generated
fragments. Sequences targeted to noncoding regions are particularly
appropriate for this use as greater numbers of polymorphisms occur
in the noncoding regions, making it easier to differentiate
individuals using this technique. Examples of polynucleotide
reagents include the nucleic acid sequences of the invention or
portions thereof, e.g., fragments derived from noncoding regions
having a length of at least 20 or 30 bases.
The nucleic acid sequences described herein can further be used to
provide polynucleotide reagents, e.g., labeled or labelable probes
which can be used in, for example, an in situ hybridization
technique, to identify a specific tissue, e.g., brain tissue. This
can be very useful in cases where a forensic pathologist is
presented with a tissue of unknown origin. Panels of such probes
can be used to identify tissue by species and/or by organ type.
C. Predictive Medicine
The present invention also pertains to the field of predictive
medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trails are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly, one aspect of the present invention
relates to diagnostic assays for determining expression of a
polypeptide or nucleic acid of the invention and/or activity of a
polypeptide of the invention, in the context of a biological sample
(e.g., blood, serum, cells, tissue) to thereby determine whether an
individual is afflicted with a disease or disorder, or is at risk
of developing a disorder, associated with aberrant expression or
activity of a polypeptide of the invention. The invention also
provides for prognostic (or predictive) assays for determining
whether an individual is at risk of developing a disorder
associated with aberrant expression or activity of a polypeptide of
the invention. For example, mutations in a gene of the invention
can be assayed in a biological sample. Such assays can be used for
prognostic or predictive purpose to thereby prophylactically treat
an individual prior to the onset of a disorder characterized by or
associated with aberrant expression or activity of a polypeptide of
the invention.
Another aspect of the invention provides methods for expression of
a nucleic acid or polypeptide of the invention or activity of a
polypeptide of the invention in an individual to thereby select
appropriate therapeutic or prophylactic agents for that individual
(referred to herein as "pharmacogenomics"). Pharmacogenomics allows
for the selection of agents (e.g., drugs) for therapeutic or
prophylactic treatment of an individual based on the genotype of
the individual (e.g., the genotype of the individual examined to
determine the ability of the individual to respond to a particular
agent).
Yet another aspect of the invention pertains to monitoring the
influence of agents (e.g., drugs or other compounds) on the
expression or activity of a polypeptide of the invention in
clinical trials. These and other agents are described in further
detail in the following sections.
1. Diagnostic Assays
An exemplary method for detecting the presence or absence of a
polypeptide or nucleic acid of the invention in a biological sample
involves obtaining a biological sample from a test subject and
contacting the biological sample with a compound or an agent
capable of detecting a polypeptide or nucleic acid (e.g., mRNA,
genomic DNA) of the invention such that the presence of a
polypeptide or nucleic acid of the invention is detected in the
biological sample. A preferred agent for detecting mRNA or genomic
DNA encoding a polypeptide of the invention is a labeled nucleic
acid probe capable of hybridizing to mRNA or genomic DNA encoding a
polypeptide of the invention. The nucleic acid probe can be, for
example, a full-length cDNA, such as the nucleic acid of SEQ ID
NO:1, 2, 3, 13, 14, 19, 20, 31, 32, 39, 40, or 44, or a portion
thereof, such as an oligonucleotide of at least 15, 30, 50, 100,
250 or 500 nucleotides in length and sufficient to specifically
hybridize under stringent conditions to a mRNA or genomic DNA
encoding a polypeptide of the invention. Other suitable probes for
use in the diagnostic assays of the invention are described
herein.
A preferred agent for detecting a polypeptide of the invention is
an antibody capable of binding to a polypeptide of the invention,
preferably an antibody with a detectable label. Antibodies can be
polyclonal, or more preferably, monoclonal. An intact antibody, or
a fragment thereof (e.g., Fab or F(ab').sub.2) can be used. The
term "labeled", with regard to the probe or antibody, is intended
to encompass direct labeling of the probe or antibody by coupling
(i.e., physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently labeled streptavidin. The term "biological sample" is
intended to include tissues, cells and biological fluids isolated
from a subject, as well as tissues, cells and fluids present within
a subject. That is, the detection method of the invention can be
used to detect mRNA, protein, or genomic DNA in a biological sample
in vitro as well as in vivo. For example, in vitro techniques for
detection of mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of a polypeptide
of the invention include enzyme linked immunosorbent assays
(ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro techniques for detection of genomic
DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of a polypeptide of the invention include
introducing into a subject a labeled antibody directed against the
polypeptide. For example, the antibody can be labeled with a
radioactive marker whose presence and location in a subject can be
detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules
from the test subject. Alternatively, the biological sample can
contain mRNA molecules from the test subject or genomic DNA
molecules from the test subject. A preferred biological sample is a
peripheral blood leukocyte sample isolated by conventional means
from a subject.
In another embodiment, the methods further involve obtaining a
control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting a
polypeptide of the invention or mRNA or genomic DNA encoding a
polypeptide of the invention, such that the presence of the
polypeptide or mRNA or genomic DNA encoding the polypeptide is
detected in the biological sample, and comparing the presence of
the polypeptide or mRNA or genomic DNA encoding the polypeptide in
the control sample with the presence of the polypeptide or mRNA or
genomic DNA encoding the polypeptide in the test sample.
The invention also encompasses kits for detecting the presence of a
polypeptide or nucleic acid of the invention in a biological sample
(a test sample). Such kits can be used to determine if a subject is
suffering from or is at increased risk of developing a disorder
associated with aberrant expression of a polypeptide of the
invention (e.g., a proliferative disorder, e.g., psoriasis or
cancer). For example, the kit can comprise a labeled compound or
agent capable of detecting the polypeptide or mRNA encoding the
polypeptide in a biological sample and means for determining the
amount of the polypeptide or mRNA in the sample (e.g., an antibody
which binds the polypeptide or an oligonucleotide probe which binds
to DNA or mRNA encoding the polypeptide). Kits can also include
instructions for observing that the tested subject is suffering
from or is at risk of developing a disorder associated with
aberrant expression of the polypeptide if the amount of the
polypeptide or mRNA encoding the polypeptide is above or below a
normal level.
For antibody-based kits, the kit can comprise, for example: (1) a
first antibody (e.g., attached to a solid support) which binds to a
polypeptide of the invention; and, optionally, (2) a second,
different antibody which binds to either the polypeptide or the
first antibody and is conjugated to a detectable agent.
For oligonucleotide-based kits, the kit can comprise, for example:
(1) an oligonucleotide, e.g., a detectably labeled oligonucleotide,
which hybridizes to a nucleic acid sequence encoding a polypeptide
of the invention or (2) a pair of primers useful for amplifying a
nucleic acid molecule encoding a polypeptide of the invention. The
kit can also comprise, e.g., a buffering agent, a preservative, or
a protein stabilizing agent. The kit can also comprise components
necessary for detecting the detectable agent (e.g., an enzyme or a
substrate). The kit can also contain a control sample or a series
of control samples which can be assayed and compared to the test
sample contained. Each component of the kit is usually enclosed
within an individual container and all of the various containers
are within a single package along with instructions for observing
whether the tested subject is suffering from or is at risk of
developing a disorder associated with aberrant expression of the
polypeptide.
2. Prognostic Assays
The methods described herein can furthermore be utilized as
diagnostic or prognostic assays to identify subjects having or at
risk of developing a disease or disorder associated with aberrant
expression or activity of a polypeptide of the invention. For
example, the assays described herein, such as the preceding
diagnostic assays or the following assays, can be utilized to
identify a subject having or at risk of developing a disorder
associated with aberrant expression or activity of a polypeptide of
the invention, e.g., an immunologic disorder, e.g., asthma,
anaphylaxis, or atopic dermatitis. Alternatively, the prognostic
assays can be utilized to identify a subject having or at risk for
developing such a disease or disorder. Thus, the present invention
provides a method in which a test sample is obtained from a subject
and a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the
invention is detected, wherein the presence of the polypeptide or
nucleic acid is diagnostic for a subject having or at risk of
developing a disease or disorder associated with aberrant
expression or activity of the polypeptide. As used herein, a "test
sample" refers to a biological sample obtained from a subject of
interest. For example, a test sample can be a biological fluid
(e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to
determine whether a subject can be administered an agent (e.g., an
agonist, antagonist, peptidomimetic, protein, peptide, nucleic
acid, small molecule, or other drug candidate) to treat a disease
or disorder associated with aberrant expression or activity of a
polypeptide of the invention. For example, such methods can be used
to determine whether a subject can be effectively treated with a
specific agent or class of agents (e.g., agents of a type which
decrease activity of the polypeptide). Thus, the present invention
provides methods for determining whether a subject can be
effectively treated with an agent for a disorder associated with
aberrant expression or activity of a polypeptide of the invention
in which a test sample is obtained and the polypeptide or nucleic
acid encoding the polypeptide is detected (e.g., wherein the
presence of the polypeptide or nucleic acid is diagnostic for a
subject that can be administered the agent to treat a disorder
associated with aberrant expression or activity of the
polypeptide).
The methods of the invention can also be used to detect genetic
lesions or mutations in a gene of the invention, thereby
determining if a subject with the lesioned gene is at risk for a
disorder characterized aberrant expression or activity of a
polypeptide of the invention. In preferred embodiments, the methods
include detecting, in a sample of cells from the subject, the
presence or absence of a genetic lesion or mutation characterized
by at least one of an alteration affecting the integrity of a gene
encoding the polypeptide of the invention, or the mis-expression of
the gene encoding the polypeptide of the invention. For example,
such genetic lesions or mutations can be detected by ascertaining
the existence of at least one of: 1) a deletion of one or more
nucleotides from the gene; 2) an addition of one or more
nucleotides to the gene; 3) a substitution of one or more
nucleotides of the gene; 4) a chromosomal rearrangement of the
gene; 5) an alteration in the level of a messenger RNA transcript
of the gene; 6) an aberrant modification of the gene, such as of
the methylation pattern of the genomic DNA; 7) the presence of a
non-wild type splicing pattern of a messenger RNA transcript of the
gene; 8) a non-wild type level of a the protein encoded by the
gene; 9) an allelic loss of the gene; and 10) an inappropriate
post-translational modification of the protein encoded by the gene.
As described herein, there are a large number of assay techniques
known in the art which can be used for detecting lesions in a
gene.
In certain embodiments, detection of the lesion involves the use of
a probe/primer in a polymerase chain reaction (PCR) (see, e.g.,
U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE
PCR, or, alternatively, in a ligation chain reaction (LCR) (see,
e.g., Landegran et al. (1988) Science 241:1077 1080; and Nakazawa
et al. (1994) Proc. Natl. Acad. Sci. USA 91:360 364), the latter of
which can be particularly useful for detecting point mutations in a
gene (see, e.g., Abravaya et al. (1995) Nucleic Acids Res. 23:675
682). This method can include the steps of collecting a sample of
cells from a patient, isolating nucleic acid (e.g., genomic, mRNA
or both) from the cells of the sample, contacting the nucleic acid
sample with one or more primers which specifically hybridize to the
selected gene under conditions such that hybridization and
amplification of the gene (if present) occurs, and detecting the
presence or absence of an amplification product, or detecting the
size of the amplification product and comparing the length to a
control sample. It is anticipated that PCR and/or LCR may be
desirable to use as a preliminary amplification step in conjunction
with any of the techniques used for detecting mutations described
herein.
Alternative amplification methods include: self sustained sequence
replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA
87:1874 1878), transcriptional amplification system (Kwoh, et al.
(1989) Proc. Natl. Acad. Sci. USA 86:1173 1177), Q-Beta Replicase
(Lizardi et al. (1988) Bio/Technology 6:1197), or any other nucleic
acid amplification method, followed by the detection of the
amplified molecules using techniques well known to those of skill
in the art. These detection schemes are especially useful for the
detection of nucleic acid molecules if such molecules are present
in very low numbers.
In an alternative embodiment, mutations in a selected gene from a
sample cell can be identified by alterations in restriction enzyme
cleavage patterns. For example, sample and control DNA is isolated,
amplified (optionally), digested with one or more restriction
endonucleases, and fragment length sizes are determined by gel
electrophoresis and compared. Differences in fragment length sizes
between sample and control DNA indicates mutations in the sample
DNA. Moreover, the use of sequence specific ribozymes (see, e.g.,
U.S. Pat. No. 5,498,531) can be used to score for the presence of
specific mutations by development or loss of a ribozyme cleavage
site.
In other embodiments, genetic mutations can be identified by
hybridizing a sample and control nucleic acids, e.g., DNA or RNA,
to high density arrays containing hundreds or thousands of
oligonucleotides probes (Cronin et al. (1996) Human Mutation 7:244
255; Kozal et al. (1996) Nature Medicine 2:753 759). For example,
genetic mutations can be identified in two-dimensional arrays
containing light-generated DNA probes as described in Cronin et
al., supra. Briefly, a first hybridization array of probes can be
used to scan through long stretches of DNA in a sample and control
to identify base changes between the sequences by making linear
arrays of sequential overlapping probes. This step allows the
identification of point mutations. This step is followed by a
second hybridization array that allows the characterization of
specific mutations by using smaller, specialized probe arrays
complementary to all variants or mutations detected. Each mutation
array is composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions
known in the art can be used to directly sequence the selected gene
and detect mutations by comparing the sequence of the sample
nucleic acids with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques
developed by Maxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA
74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It
is also contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
((1995) Bio/Techniques 19:448), including sequencing by mass
spectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et
al. (1996) Adv. Chromatogr. 36:127 162; and Griffin et al. (1993)
Appl. Biochem. Biotechnol. 38:147 159).
Other methods for detecting mutations in a selected gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.
(1985) Science 230:1242). In general, the technique of "mismatch
cleavage" entails providing heteroduplexes formed by hybridizing
(labeled) RNA or DNA containing the wild-type sequence with
potentially mutant RNA or DNA obtained from a tissue sample. The
double-stranded duplexes are treated with an agent which cleaves
single-stranded regions of the duplex such as which will exist due
to basepair mismatches between the control and sample strands.
RNA/DNA duplexes can be treated with RNase to digest mismatched
regions, and DNA/DNA hybrids can be treated with S1 nuclease to
digest mismatched regions.
In other embodiments, either DNA/DNA or RNA/DNA duplexes can be
treated with hydroxylamine or osmium tetroxide and with piperidine
in order to digest mismatched regions. After digestion of the
mismatched regions, the resulting material is then separated by
size on denaturing polyacrylamide gels to determine the site of
mutation. See, e.g., Cotton et al. (1988) Proc. Natl. Acad. Sci.
USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286 295. In
a preferred embodiment, the control DNA or RNA can be labeled for
detection.
In still another embodiment, the mismatch cleavage reaction employs
one or more proteins that recognize mismatched base pairs in
double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in cDNAs
obtained from samples of cells. For example, the mutY enzyme of E.
coli cleaves A at G/A mismatches and the thymidine DNA glycosylase
from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994)
Carcinogenesis 15:1657 1662). According to an exemplary embodiment,
a probe based on a selected sequence, e.g., a wild-type sequence,
is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is treated with a DNA mismatch repair enzyme, and the
cleavage products, if any, can be detected from electrophoresis
protocols or the like. See, e.g., U.S. Pat. No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will
be used to identify mutations in genes. For example, single strand
conformation polymorphism (SSCP) may be used to detect differences
in electrophoretic mobility between mutant and wild type nucleic
acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see
also Cotton (1993) Mutat. Res. 285:125 144; Hayashi (1992) Genet.
Anal. Tech. Appl. 9:73 79). Single-stranded DNA fragments of sample
and control nucleic acids will be denatured and allowed to
renature. The secondary structure of single-stranded nucleic acids
varies according to sequence, and the resulting alteration in
electrophoretic mobility enables the detection of even a single
base change. The DNA fragments may be labeled or detected with
labeled probes. The sensitivity of the assay may be enhanced by
using RNA (rather than DNA), in which the secondary structure is
more sensitive to a change in sequence. In a preferred embodiment,
the subject method utilizes heteroduplex analysis to separate
double stranded heteroduplex molecules on the basis of changes in
electrophoretic mobility (Keen et al. (1991) Trends Genet.
7:5).
In yet another embodiment, the movement of mutant or wild-type
fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys.
Chem. 265:12753).
Examples of other techniques for detecting point mutations include,
but are not limited to, selective oligonucleotide hybridization,
selective amplification, or selective primer extension. For
example, oligonucleotide primers may be prepared in which the known
mutation is placed centrally and then hybridized to target DNA
under conditions which permit hybridization only if a perfect match
is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989)
Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific
oligonucleotides are hybridized to PCR amplified target DNA or a
number of different mutations when the oligonucleotides are
attached to the hybridizing membrane and hybridized with labeled
target DNA.
Alternatively, allele specific amplification technology which
depends on selective PCR amplification may be used in conjunction
with the instant invention. Oligonucleotides used as primers for
specific amplification may carry the mutation of interest in the
center of the molecule (so that amplification depends on
differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res.
17:2437 2448) or at the extreme 3' end of one primer where, under
appropriate conditions, mismatch can prevent or reduce polymerase
extension (Prossner (1993) Tibtech 11:238). In addition, it may be
desirable to introduce a novel restriction site in the region of
the mutation to create cleavage-based detection (Gasparini et al.
(1992) Mol. Cell Probes 6:1). It is anticipated that in certain
embodiments amplification may also be performed using Taq ligase
for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA
88:189). In such cases, ligation will occur only if there is a
perfect match at the 3' end of the 5' sequence making it possible
to detect the presence of a known mutation at a specific site by
looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by
utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a gene encoding a polypeptide of the invention.
Furthermore, any cell type or tissue, e.g., chondrocytes, in which
the polypeptide of the invention is expressed may be utilized in
the prognostic assays described herein.
3. Pharmacogenomics
Agents, or modulators which have a stimulatory or inhibitory effect
on activity or expression of a polypeptide of the invention as
identified by a screening assay described herein can be
administered to individuals to treat (prophylactically or
therapeutically) disorders associated with aberrant activity of the
polypeptide. In conjunction with such treatment, the
pharmacogenomics (i.e., the study of the relationship between an
individual's genotype and that individual's response to a foreign
compound or drug) of the individual may be considered. Differences
in metabolism of therapeutics can lead to severe toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus, the
pharmacogenomics of the individual permits the selection of
effective agents (e.g., drugs) for prophylactic or therapeutic
treatments based on a consideration of the individual's genotype.
Such pharmacogenomics can further be used to determine appropriate
dosages and therapeutic regimens. Accordingly, the activity of a
polypeptide of the invention, expression of a nucleic acid of the
invention, or mutation content of a gene of the invention in an
individual can be determined to thereby select appropriate agent(s)
for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Linder (1997)
Clin. Chem. 43(2):254 266. In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body are referred
to as "altered drug action." Genetic conditions transmitted as
single factors altering the way the body acts on drugs are referred
to as "altered drug metabolism". These pharmacogenetic conditions
can occur either as rare defects or as polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
As an illustrative embodiment, the activity of drug metabolizing
enzymes is a major determinant of both the intensity and duration
of drug action. The discovery of genetic polymorphisms of drug
metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and
cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, a PM will show no therapeutic
response, as demonstrated for the analgesic effect of codeine
mediated by its CYP2D6-formed metabolite morphine. The other
extreme are the so called ultra-rapid metabolizers who do not
respond to standard doses. Recently, the molecular basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
Thus, the activity of a polypeptide of the invention, expression of
a nucleic acid encoding the polypeptide, or mutation content of a
gene encoding the polypeptide in an individual can be determined to
thereby select appropriate agent(s) for therapeutic or prophylactic
treatment of the individual. In addition, pharmacogenetic studies
can be used to apply genotyping of polymorphic alleles encoding
drug-metabolizing enzymes to the identification of an individual's
drug responsiveness phenotype. This knowledge, when applied to
dosing or drug selection, can avoid adverse reactions or
therapeutic failure and thus enhance therapeutic or prophylactic
efficiency when treating a subject with a modulator of activity or
expression of the polypeptide, such as a modulator identified by
one of the exemplary screening assays described herein.
4. Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the
expression or activity of a polypeptide of the invention (e.g., the
ability to modulate aberrant cell proliferation chemotaxis, and/or
differentiation) can be applied not only in basic drug screening,
but also in clinical trials. For example, the effectiveness of an
agent, as determined by a screening assay as described herein, to
increase gene expression, protein levels or protein activity, can
be monitored in clinical trials of subjects exhibiting decreased
gene expression, protein levels, or protein activity.
Alternatively, the effectiveness of an agent, as determined by a
screening assay, to decrease gene expression, protein levels or
protein activity, can be monitored in clinical trials of subjects
exhibiting increased gene expression, protein levels, or protein
activity. In such clinical trials, expression or activity of a
polypeptide of the invention and preferably, that of other
polypeptide that have been implicated in for example, a cellular
proliferation disorder, can be used as a marker of the immune
responsiveness of a particular cell.
For example, and not by way of limitation, genes, including those
of the invention, that are modulated in cells by treatment with an
agent (e.g., compound, drug or small molecule) which modulates
activity or expression of a polypeptide of the invention (e.g., as
identified in a screening assay described herein) can be
identified. Thus, to study the effect of agents on cellular
proliferation disorders, for example, in a clinical trial, cells
can be isolated and RNA prepared and analyzed for the levels of
expression of a gene of the invention and other genes implicated in
the disorder. The levels of gene expression (i.e., a gene
expression pattern) can be quantified by Northern blot analysis or
RT-PCR, as described herein, or alternatively by measuring the
amount of protein produced, by one of the methods as described
herein, or by measuring the levels of activity of a gene of the
invention or other genes. In this way, the gene expression pattern
can serve as a marker, indicative of the physiological response of
the cells to the agent. Accordingly, this response state may be
determined before, and at various points during, treatment of the
individual with the agent.
In a preferred embodiment, the present invention provides a method
for monitoring the effectiveness of treatment of a subject with an
agent (e.g., an agonist, antagonist, peptidomimetic, protein,
peptide, nucleic acid, small molecule, or other drug candidate
identified by the screening assays described herein) comprising the
steps of (i) obtaining a pre-administration sample from a subject
prior to administration of the agent; (ii) detecting the level of
the polypeptide or nucleic acid of the invention in the
preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level the of the polypeptide or nucleic acid of the invention in
the post-administration samples; (v) comparing the level of the
polypeptide or nucleic acid of the invention in the
pre-administration sample with the level of the polypeptide or
nucleic acid of the invention in the post-administration sample or
samples; and (vi) altering the administration of the agent to the
subject accordingly. For example, increased administration of the
agent may be desirable to increase the expression or activity of
the polypeptide to higher levels than detected, i.e., to increase
the effectiveness of the agent. Alternatively, decreased
administration of the agent may be desirable to decrease expression
or activity of the polypeptide to lower levels than detected, i.e.,
to decrease the effectiveness of the agent.
C. Methods of Treatment
The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant expression or activity of a polypeptide of the invention.
For example, disorders characterized by aberrant expression or
activity of the polypeptides of the invention include immunologic
disorders. In addition, the nucleic acids, polypeptides, and
modulators thereof of the invention can be used to treat
immunologic diseases and disorders, including but not limited to,
allergic disorders (e.g., anaphylaxis and allergic asthma) and
inflammatory disorders (e.g., atopic dermatitis). Polypeptides of
the invention can also treat diseases associated with bacterial
infection (e.g., tuberculosis, e.g., pulmonary tuberculosis),
inflammatory arthropathy, and bone and cartilage degenerative
diseases and disorders (e.g., arthritis, e.g., rheumatoid
arthritis), as well as other disorders described herein.
1. Prophylactic Methods
In one aspect, the invention provides a method for preventing in a
subject, a disease or condition associated with an aberrant
expression or activity of a polypeptide of the invention, by
administering to the subject an agent which modulates expression or
at least one activity of the polypeptide. Subjects at risk for a
disease which is caused or contributed to by aberrant expression or
activity of a polypeptide of the invention can be identified by,
for example, any or a combination of diagnostic or prognostic
assays as described herein. Administration of a prophylactic agent
can occur prior to the manifestation of symptoms characteristic of
the aberrancy, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on the type of
aberrancy, for example, an agonist or antagonist agent can be used
for treating the subject. For example, an antagonist of a TANGO 240
protein may be used to treat an arthropathic disorder, e.g.,
rheumatoid arthritis. The appropriate agent can be determined based
on screening assays described herein.
2. Therapeutic Methods
Another aspect of the invention pertains to methods of modulating
expression or activity of a polypeptide of the invention for
therapeutic purposes. The modulatory method of the invention
involves contacting a cell with an agent that modulates one or more
of the activities of the polypeptide. An agent that modulates
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring cognate ligand of the
polypeptide, a peptide, a peptidomimetic, or other small molecule.
In one embodiment, the agent stimulates one or more of the
biological activities of the polypeptide. Examples of such
stimulatory agents include the active polypeptide of the invention
and a nucleic acid molecule encoding the polypeptide of the
invention that has been introduced into the cell. In another
embodiment, the agent inhibits one or more of the biological
activities of the polypeptide of the invention. Examples of such
inhibitory agents include antisense nucleic acid molecules and
antibodies. These modulatory methods can be performed in vitro
(e.g., by culturing the cell with the agent) or, alternatively, in
vivo (e.g., by administering the agent to a subject). As such, the
present invention provides methods of treating an individual
afflicted with a disease or disorder characterized by aberrant
expression or activity of a polypeptide of the invention. In one
embodiment, the method involves administering an agent (e.g., an
agent identified by a screening assay described herein), or
combination of agents that modulates (e.g., upregulates or
downregulates) expression or activity. In another embodiment, the
method involves administering a polypeptide of the invention or a
nucleic acid molecule of the invention as therapy to compensate for
reduced or aberrant expression or activity of the polypeptide.
Stimulation of activity is desirable in situations in which
activity or expression is abnormally low or downregulated and/or in
which increased activity is likely to have a beneficial effect.
Conversely, inhibition of activity is desirable in situations in
which activity or expression is abnormally high or upregulated
and/or in which decreased activity is likely to have a beneficial
effect.
This invention is further illustrated by the following examples
which should not be construed as limiting. The contents of all
references, patents and published patent applications cited
throughout this application are hereby incorporated by
reference.
Deposit of Clones
Clones containing cDNA molecules encoding TANGO 228, (clone
EpT228), TANGO 240 (EpT240) and TANGO 243 (clone EpT243) were
deposited with the American Type Culture Collection, 10801
University Boulevard, Manassas, Va., 20110-2209, on Feb. 18, 1999
as Accession Number 207116, as part of a composite deposit
representing a mixture of three strains, each carrying one
recombinant plasmid harboring a particular cDNA clone.
To distinguish the strains and isolate a strain harboring a
particular cDNA clone, an aliquot of the mixture can be streaked
out to single colonies on nutrient medium (e.g., LB plates)
supplemented with 100 .mu.g/ml ampicillin, single colonies grown,
and then plasmid DNA extracted using a standard minipreparation
procedure. Next, a sample of the DNA minipreparation can be
digested with a combination of the restriction enzymes Sal I and
Not I and the resultant products resolved on a 0.8% agarose gel
using standard DNA electrophoresis conditions. The digest liberates
fragments as follows:
TANGO 228: 4.1 kb
TANGO 240: 2.2 kb
TANGO 243: 2.8 kb
The identity of the strains can be inferred from the fragments
liberated.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following
claims.
SEQUENCE LISTINGS
1
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 51 <210>
SEQ ID NO 1 <211> LENGTH: 4043 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (34)...(1062) <400>
SEQUENCE: 1 cttctgaccc cgtcttggac ttcaactggg aga atg tgg agc cat
ttg agc agg 54 Met Trp Ser His Leu Ser Arg 1 5 ctc ctc ttc tgg agc
ata ttt tct tct gtc act tgt aga aaa gct gta 102 Leu Leu Phe Trp Ser
Ile Phe Ser Ser Val Thr Cys Arg Lys Ala Val 10 15 20 ttg gat tgt
gag gca atg aaa aca aat gaa ttc cct tct cca tgt ttg 150 Leu Asp Cys
Glu Ala Met Lys Thr Asn Glu Phe Pro Ser Pro Cys Leu 25 30 35 gac
tca aag act aag gtg gtt atg aag ggt caa aat gta tct atg ttt 198 Asp
Ser Lys Thr Lys Val Val Met Lys Gly Gln Asn Val Ser Met Phe 40 45
50 55 tgt tcc cat aag aac aaa tca ctg cag atc acc tat tca ttg ttt
cga 246 Cys Ser His Lys Asn Lys Ser Leu Gln Ile Thr Tyr Ser Leu Phe
Arg 60 65 70 cgt aag aca cac ctg gga acc cag gat gga aaa ggt gaa
cct gcg att 294 Arg Lys Thr His Leu Gly Thr Gln Asp Gly Lys Gly Glu
Pro Ala Ile 75 80 85 ttt aac cta agc atc aca gaa gcc cat gaa tca
ggc ccc tac aaa tgc 342 Phe Asn Leu Ser Ile Thr Glu Ala His Glu Ser
Gly Pro Tyr Lys Cys 90 95 100 aaa gcc caa gtt acc agc tgt tca aaa
tac agt cgt gac ttc agc ttc 390 Lys Ala Gln Val Thr Ser Cys Ser Lys
Tyr Ser Arg Asp Phe Ser Phe 105 110 115 acg att gtc gac ccg gtg act
tcc cca gtg ctg aac att atg gtc att 438 Thr Ile Val Asp Pro Val Thr
Ser Pro Val Leu Asn Ile Met Val Ile 120 125 130 135 caa aca gaa aca
gac cga cat ata aca tta cat tgc ctc tca gtc aat 486 Gln Thr Glu Thr
Asp Arg His Ile Thr Leu His Cys Leu Ser Val Asn 140 145 150 ggc tcg
ctg ccc atc aat tac act ttc ttt gaa aac cat gtt gcc ata 534 Gly Ser
Leu Pro Ile Asn Tyr Thr Phe Phe Glu Asn His Val Ala Ile 155 160 165
tca cca gct att tcc aag tat gac agg gag cct gct gaa ttt aac tta 582
Ser Pro Ala Ile Ser Lys Tyr Asp Arg Glu Pro Ala Glu Phe Asn Leu 170
175 180 acc aag aag aat cct gga gaa gag gaa gag tat agg tgt gaa gct
aaa 630 Thr Lys Lys Asn Pro Gly Glu Glu Glu Glu Tyr Arg Cys Glu Ala
Lys 185 190 195 aac aga ttg cct aac tat gca aca tac agt cac cct gtc
acc atg ccc 678 Asn Arg Leu Pro Asn Tyr Ala Thr Tyr Ser His Pro Val
Thr Met Pro 200 205 210 215 tca aca ggc gga gac agc tgt cct ttc tgt
ctg aag cta cta ctt cca 726 Ser Thr Gly Gly Asp Ser Cys Pro Phe Cys
Leu Lys Leu Leu Leu Pro 220 225 230 ggg tta tta ctg ttg ctg gtg gtg
ata atc cta att ctg gct ttt tgg 774 Gly Leu Leu Leu Leu Leu Val Val
Ile Ile Leu Ile Leu Ala Phe Trp 235 240 245 gta ctg ccc aaa tac aaa
aca aga aaa gct atg aga aat aat gtg ccc 822 Val Leu Pro Lys Tyr Lys
Thr Arg Lys Ala Met Arg Asn Asn Val Pro 250 255 260 agg gac cgt gga
gac aca gcc atg gaa gtt gga atc tat gca aat atc 870 Arg Asp Arg Gly
Asp Thr Ala Met Glu Val Gly Ile Tyr Ala Asn Ile 265 270 275 ctt gaa
aaa caa gca aag gag gaa tct gtg cca gaa gtg gga tcc agg 918 Leu Glu
Lys Gln Ala Lys Glu Glu Ser Val Pro Glu Val Gly Ser Arg 280 285 290
295 ccg tgt gtt tcc aca gcc caa gat gag gcc aaa cac tcc cag gag cta
966 Pro Cys Val Ser Thr Ala Gln Asp Glu Ala Lys His Ser Gln Glu Leu
300 305 310 cag tat gcc acc ccc gtg ttc cag gag gtg gca cca aga gag
caa gaa 1014 Gln Tyr Ala Thr Pro Val Phe Gln Glu Val Ala Pro Arg
Glu Gln Glu 315 320 325 gcc tgt gat tct tat aaa tct gga tat gtc tat
tct gaa ctc aac ttc 1062 Ala Cys Asp Ser Tyr Lys Ser Gly Tyr Val
Tyr Ser Glu Leu Asn Phe 330 335 340 tgaaatttac agaaacaaac
tacatctcag gtagagacag ggttttgcca tgttggccag 1122 gctggtcttc
aactcctgac ctcaagtgat ccgcccacct cggactccca gggtgctggg 1182
attacaggcg tgagccacct cgcctggccc tccatttcct gatctagtct tatatccacg
1242 ctcaccacct cagcacgctc agacccacgc tgctgtgggc tcctctggct
cctggaagag 1302 tgcgtccgca gatgctgcag tctttgtgtg gctcagcaat
tgccactcac atcaggaact 1362 gcctttaccc tgtcaggctc tactgagacc
cgaccctggt tattaagcta taggggagac 1422 aaggatggat cttaaagaag
acaagcaaaa tatagtgaag caaatagaat ggtggttcct 1482 gggggatggg
ggcaggggac aacgaggagt gactggctaa cagatacagc gtttcagttt 1542
ggaaagacaa aaaagttctg gaaaaaagat ggaaggtggt gatggttgca caataatatg
1602 agttttgttg ttgttgtttt ttgagacagg gtctctctct gttgcccagg
ctggagtgca 1662 ggggcacgat ttcggctcac tgcaacctct gcctcccggg
ttcgggcgat tcttgtgcct 1722 cagcctccca ggtagctggg attgcaggcg
cccaccaccg tgcccagcta atttttgtat 1782 ttttagtggg gatggggttt
caccatgttg gccaggctgg tctcgaactc ctgacctcaa 1842 gtgattggcc
tgccttggcc tcccaaagtg ctgggattac aggcatgagc tattgcgccc 1902
ggcaatatga atgaatgttt ttaataccat tgaattacac acctaaaatt ggttaaaatg
1962 gtaaaaaatt ttatgttatg tataacttac cacaataatt taaaaaatat
tgtgaagcgc 2022 ctgcctcatg tgaggtcttc taataagagg ggctgttctc
cttctcaaga gcgctcgcgg 2082 gcattgggag tttcttattc tcaaatgtcc
acatcccaag gcctaccccg tccctgtcag 2142 tgtcagaagt tagcagagca
gaactgctag atgtgctcac tccatctcct ccatggttgg 2202 cccacccttt
cgatcagatc ctggaattgg ctttcagcac agccttccag ctggctgtga 2262
taaatgagag tggcttaaac cctgtcatca gggtttcaca gcttgccctg aaccgtagct
2322 tctcattttc tccttgcaaa tcttctaagg cagctaagag aagccaatta
acttcacaat 2382 ccacataatt agcattgccc caaacctttc aagtgctgga
atgctggcgt ccactagtgc 2442 ctcgtttctt tctaaacctc attccacatg
gcaggggaag gtcttaggaa ttgtggagct 2502 gtggcgttct aagggttctc
actgcctacc tatcaccagc aagggagtcc ttgttgccat 2562 ccatccctag
ggggtaattt tgttccctga ggctgctttc tagggacttc tggtcgcttg 2622
ttttatcctg gaccagacct gaaagcagag cctgaaataa ggccttctat gcacatcatt
2682 tatgtaggag gtggccctag gaagcaggcc caatgcgcca tgggaaaaac
cagtaccagg 2742 gtgttttgct gagttgagca ctgtggtggg cagctggaca
tgagcccact ggaatcttct 2802 gaagagccca agagcctctt ctcagtattg
tccacttgag gattgatagt gagaggcatt 2862 tatccactgg tgtccaacgc
tcactggttg aggatcaccc cagaaagcga cacctccccc 2922 acttctagac
tagcatgtgg gtattccaag caggcttacc tcagtgtctc accccaggca 2982
ctgcaaacac cccaggacag aaagtgaaca tgtgtattgt gcaattgaag caagacacta
3042 tcagtgcaaa gtgagtagac cctcagatgc tgctgggtca gagggaaggc
ccagggatat 3102 gacacaggac acagaggtgg cagatacacc acctcccaac
atttcctcta tcacagcaac 3162 tagaacaatg gcaacaatag gggtgggtgt
ggtggctcgc gcctgtggtc ccaataccct 3222 gggaggccag ggcagaagga
ttgcttgagc ccaggagttc aagactagcc tgggcaactt 3282 ggcgaaaccc
tgtctctaca aacatatttg tatgtgattg aacaagtaga acaatggaac 3342
ggaaagtcca gatgtagttc taaatatgta caggaactta gtataggata aatatggcat
3402 cttaaatcaa tggggaaaag atggattatt caacatattg taaaaacttg
agaatcacat 3462 taaaaaagtt ggactcctac ctcattcttt acaacaaaat
taattctgat agatgtagtc 3522 acagaagagc tagaagaata tgcaggggat
ttaaaaataa tcttgaagtg gggaggactt 3582 ttataggtat gactcacaac
ccagaaaaca taaaggattg ttaaatttaa ttagataaat 3642 catttaatta
gatgaaacat ttaaaaatta catttagaaa tcataaacta aatccaaaga 3702
ataactaggg gccgggcgca gtggctcacg tctgtaatcc catggggcag gtggatcacc
3762 tgaggttggg agtttgggac cagcctgtcc agcacggtga ggccccgtct
ctactaaaaa 3822 tacaaaaatt agttggtgtg gtggcacata cctgtggtcc
ctgctactgg ggaggctgag 3882 gcatgagaat tgcttgaacc tgggaggtgg
aggttgcagt gagccaagat cgcgccactg 3942 cattccagcc tgggcgacag
agcaagactc catctcaaaa aaaaaaaaaa aaaaaaaaaa 4002 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 4043 <210> SEQ ID NO 2
<211> LENGTH: 1029 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 2 atgtggagcc
atttgagcag gctcctcttc tggagcatat tttcttctgt cacttgtaga 60
aaagctgtat tggattgtga ggcaatgaaa acaaatgaat tcccttctcc atgtttggac
120 tcaaagacta aggtggttat gaagggtcaa aatgtatcta tgttttgttc
ccataagaac 180 aaatcactgc agatcaccta ttcattgttt cgacgtaaga
cacacctggg aacccaggat 240 ggaaaaggtg aacctgcgat ttttaaccta
agcatcacag aagcccatga atcaggcccc 300 tacaaatgca aagcccaagt
taccagctgt tcaaaataca gtcgtgactt cagcttcacg 360 attgtcgacc
cggtgacttc cccagtgctg aacattatgg tcattcaaac agaaacagac 420
cgacatataa cattacattg cctctcagtc aatggctcgc tgcccatcaa ttacactttc
480 tttgaaaacc atgttgccat atcaccagct atttccaagt atgacaggga
gcctgctgaa 540 tttaacttaa ccaagaagaa tcctggagaa gaggaagagt
ataggtgtga agctaaaaac 600 agattgccta actatgcaac atacagtcac
cctgtcacca tgccctcaac aggcggagac 660 agctgtcctt tctgtctgaa
gctactactt ccagggttat tactgttgct ggtggtgata 720 atcctaattc
tggctttttg ggtactgccc aaatacaaaa caagaaaagc tatgagaaat 780
aatgtgccca gggaccgtgg agacacagcc atggaagttg gaatctatgc aaatatcctt
840 gaaaaacaag caaaggagga atctgtgcca gaagtgggat ccaggccgtg
tgtttccaca 900 gcccaagatg aggccaaaca ctcccaggag ctacagtatg
ccacccccgt gttccaggag 960 gtggcaccaa gagagcaaga agcctgtgat
tcttataaat ctggatatgt ctattctgaa 1020 ctcaacttc 1029 <210>
SEQ ID NO 3
<211> LENGTH: 343 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY: SIGNAL
<222> LOCATION: (1)...(19) <400> SEQUENCE: 3 Met Trp
Ser His Leu Ser Arg Leu Leu Phe Trp Ser Ile Phe Ser Ser -15 -10 -5
Val Thr Cys Arg Lys Ala Val Leu Asp Cys Glu Ala Met Lys Thr Asn 1 5
10 Glu Phe Pro Ser Pro Cys Leu Asp Ser Lys Thr Lys Val Val Met Lys
15 20 25 Gly Gln Asn Val Ser Met Phe Cys Ser His Lys Asn Lys Ser
Leu Gln 30 35 40 45 Ile Thr Tyr Ser Leu Phe Arg Arg Lys Thr His Leu
Gly Thr Gln Asp 50 55 60 Gly Lys Gly Glu Pro Ala Ile Phe Asn Leu
Ser Ile Thr Glu Ala His 65 70 75 Glu Ser Gly Pro Tyr Lys Cys Lys
Ala Gln Val Thr Ser Cys Ser Lys 80 85 90 Tyr Ser Arg Asp Phe Ser
Phe Thr Ile Val Asp Pro Val Thr Ser Pro 95 100 105 Val Leu Asn Ile
Met Val Ile Gln Thr Glu Thr Asp Arg His Ile Thr 110 115 120 125 Leu
His Cys Leu Ser Val Asn Gly Ser Leu Pro Ile Asn Tyr Thr Phe 130 135
140 Phe Glu Asn His Val Ala Ile Ser Pro Ala Ile Ser Lys Tyr Asp Arg
145 150 155 Glu Pro Ala Glu Phe Asn Leu Thr Lys Lys Asn Pro Gly Glu
Glu Glu 160 165 170 Glu Tyr Arg Cys Glu Ala Lys Asn Arg Leu Pro Asn
Tyr Ala Thr Tyr 175 180 185 Ser His Pro Val Thr Met Pro Ser Thr Gly
Gly Asp Ser Cys Pro Phe 190 195 200 205 Cys Leu Lys Leu Leu Leu Pro
Gly Leu Leu Leu Leu Leu Val Val Ile 210 215 220 Ile Leu Ile Leu Ala
Phe Trp Val Leu Pro Lys Tyr Lys Thr Arg Lys 225 230 235 Ala Met Arg
Asn Asn Val Pro Arg Asp Arg Gly Asp Thr Ala Met Glu 240 245 250 Val
Gly Ile Tyr Ala Asn Ile Leu Glu Lys Gln Ala Lys Glu Glu Ser 255 260
265 Val Pro Glu Val Gly Ser Arg Pro Cys Val Ser Thr Ala Gln Asp Glu
270 275 280 285 Ala Lys His Ser Gln Glu Leu Gln Tyr Ala Thr Pro Val
Phe Gln Glu 290 295 300 Val Ala Pro Arg Glu Gln Glu Ala Cys Asp Ser
Tyr Lys Ser Gly Tyr 305 310 315 Val Tyr Ser Glu Leu Asn Phe 320
<210> SEQ ID NO 4 <211> LENGTH: 324 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Arg
Lys Ala Val Leu Asp Cys Glu Ala Met Lys Thr Asn Glu Phe Pro 1 5 10
15 Ser Pro Cys Leu Asp Ser Lys Thr Lys Val Val Met Lys Gly Gln Asn
20 25 30 Val Ser Met Phe Cys Ser His Lys Asn Lys Ser Leu Gln Ile
Thr Tyr 35 40 45 Ser Leu Phe Arg Arg Lys Thr His Leu Gly Thr Gln
Asp Gly Lys Gly 50 55 60 Glu Pro Ala Ile Phe Asn Leu Ser Ile Thr
Glu Ala His Glu Ser Gly 65 70 75 80 Pro Tyr Lys Cys Lys Ala Gln Val
Thr Ser Cys Ser Lys Tyr Ser Arg 85 90 95 Asp Phe Ser Phe Thr Ile
Val Asp Pro Val Thr Ser Pro Val Leu Asn 100 105 110 Ile Met Val Ile
Gln Thr Glu Thr Asp Arg His Ile Thr Leu His Cys 115 120 125 Leu Ser
Val Asn Gly Ser Leu Pro Ile Asn Tyr Thr Phe Phe Glu Asn 130 135 140
His Val Ala Ile Ser Pro Ala Ile Ser Lys Tyr Asp Arg Glu Pro Ala 145
150 155 160 Glu Phe Asn Leu Thr Lys Lys Asn Pro Gly Glu Glu Glu Glu
Tyr Arg 165 170 175 Cys Glu Ala Lys Asn Arg Leu Pro Asn Tyr Ala Thr
Tyr Ser His Pro 180 185 190 Val Thr Met Pro Ser Thr Gly Gly Asp Ser
Cys Pro Phe Cys Leu Lys 195 200 205 Leu Leu Leu Pro Gly Leu Leu Leu
Leu Leu Val Val Ile Ile Leu Ile 210 215 220 Leu Ala Phe Trp Val Leu
Pro Lys Tyr Lys Thr Arg Lys Ala Met Arg 225 230 235 240 Asn Asn Val
Pro Arg Asp Arg Gly Asp Thr Ala Met Glu Val Gly Ile 245 250 255 Tyr
Ala Asn Ile Leu Glu Lys Gln Ala Lys Glu Glu Ser Val Pro Glu 260 265
270 Val Gly Ser Arg Pro Cys Val Ser Thr Ala Gln Asp Glu Ala Lys His
275 280 285 Ser Gln Glu Leu Gln Tyr Ala Thr Pro Val Phe Gln Glu Val
Ala Pro 290 295 300 Arg Glu Gln Glu Ala Cys Asp Ser Tyr Lys Ser Gly
Tyr Val Tyr Ser 305 310 315 320 Glu Leu Asn Phe <210> SEQ ID
NO 5 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
SIGNAL <222> LOCATION: (1)...(19) <400> SEQUENCE: 5 Met
Trp Ser His Leu Ser Arg Leu Leu Phe Trp Ser Ile Phe Ser Ser -15 -10
-5 Val Thr Cys <210> SEQ ID NO 6 <211> LENGTH: 227
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 6 Met Trp Ser His Leu Ser Arg Leu Leu Phe Trp
Ser Ile Phe Ser Ser 1 5 10 15 Val Thr Cys Arg Lys Ala Val Leu Asp
Cys Glu Ala Met Lys Thr Asn 20 25 30 Glu Phe Pro Ser Pro Cys Leu
Asp Ser Lys Thr Lys Val Val Met Lys 35 40 45 Gly Gln Asn Val Ser
Met Phe Cys Ser His Lys Asn Lys Ser Leu Gln 50 55 60 Ile Thr Tyr
Ser Leu Phe Arg Arg Lys Thr His Leu Gly Thr Gln Asp 65 70 75 80 Gly
Lys Gly Glu Pro Ala Ile Phe Asn Leu Ser Ile Thr Glu Ala His 85 90
95 Glu Ser Gly Pro Tyr Lys Cys Lys Ala Gln Val Thr Ser Cys Ser Lys
100 105 110 Tyr Ser Arg Asp Phe Ser Phe Thr Ile Val Asp Pro Val Thr
Ser Pro 115 120 125 Val Leu Asn Ile Met Val Ile Gln Thr Glu Thr Asp
Arg His Ile Thr 130 135 140 Leu His Cys Leu Ser Val Asn Gly Ser Leu
Pro Ile Asn Tyr Thr Phe 145 150 155 160 Phe Glu Asn His Val Ala Ile
Ser Pro Ala Ile Ser Lys Tyr Asp Arg 165 170 175 Glu Pro Ala Glu Phe
Asn Leu Thr Lys Lys Asn Pro Gly Glu Glu Glu 180 185 190 Glu Tyr Arg
Cys Glu Ala Lys Asn Arg Leu Pro Asn Tyr Ala Thr Tyr 195 200 205 Ser
His Pro Val Thr Met Pro Ser Thr Gly Gly Asp Ser Cys Pro Phe 210 215
220 Cys Leu Lys 225 <210> SEQ ID NO 7 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 7 Leu Leu Leu Pro Gly Leu Leu Leu Leu Leu Val
Val Ile Ile Leu Ile 1 5 10 15 Leu Ala Phe Trp Val Leu 20
<210> SEQ ID NO 8 <211> LENGTH: 57 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 8 Gly
Gln Asn Val Ser Met Phe Cys Ser His Lys Asn Lys Ser Leu Gln 1 5 10
15 Ile Thr Tyr Ser Leu Phe Arg Arg Lys Thr His Leu Gly Thr Gln Asp
20 25 30 Gly Lys Gly Glu Pro Ala Ile Phe Asn Leu Ser Ile Thr Glu
Ala His 35 40 45 Glu Ser Gly Pro Tyr Lys Cys Lys Ala 50 55
<210> SEQ ID NO 9 <211> LENGTH: 59
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 9 Asp Arg His Ile Thr Leu His Cys Leu Ser Val
Asn Gly Ser Leu Pro 1 5 10 15 Ile Asn Tyr Thr Phe Phe Glu Asn His
Val Ala Ile Ser Pro Ala Ile 20 25 30 Ser Lys Tyr Asp Arg Glu Pro
Ala Glu Phe Asn Leu Thr Lys Lys Asn 35 40 45 Pro Gly Glu Glu Glu
Glu Tyr Arg Cys Glu Ala 50 55 <210> SEQ ID NO 10 <211>
LENGTH: 94 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 10 Pro Lys Tyr Lys Thr Arg Lys Ala Met Arg
Asn Asn Val Pro Arg Asp 1 5 10 15 Arg Gly Asp Thr Ala Met Glu Val
Gly Ile Tyr Ala Asn Ile Leu Glu 20 25 30 Lys Gln Ala Lys Glu Glu
Ser Val Pro Glu Val Gly Ser Arg Pro Cys 35 40 45 Val Ser Thr Ala
Gln Asp Glu Ala Lys His Ser Gln Glu Leu Gln Tyr 50 55 60 Ala Thr
Pro Val Phe Gln Glu Val Ala Pro Arg Glu Gln Glu Ala Cys 65 70 75 80
Asp Ser Tyr Lys Ser Gly Tyr Val Tyr Ser Glu Leu Asn Phe 85 90
<210> SEQ ID NO 11 <211> LENGTH: 1659 <212> TYPE:
DNA <213> ORGANISM: Rattus norvegicus <400> SEQUENCE:
11 ctaaatcctc tcatttcctg catgtggttg ggaagaggaa tcttagaaga
cgagccccac 60 tctgtagcag ctcaactaac catgggcgat gatgacaccc
ctgtgtgcct ctctgtcgcc 120 tcatgcaagg gagtgagttg ctggctggac
aaactcttac tctgggctct tactctgtct 180 atcacacttc gaaacaccgc
cgtggattgt aggagggtgg acagaaatgg attgctttct 240 ccaaatctga
actcaagtat gagtgtggtc aggatgggcc aaaatgtatc tctgtcttgt 300
tccagcaaga acacatccat agacatcacc tattcgctct ttttgggtaa gagataccta
360 gaaagcaaga ggagaagagg gggagctgtg gatttccacc tgaggatctc
caatgccaac 420 gagtcaggcc cctacaagtg caaagtcaat gattccaact
cgtcgaaata cagtcagaat 480 ttcaacttca caatcatcca ggatgagagc
tgctcttctt gtctactgtc gctgttgctc 540 ccaggggtgt tattggggct
aatactccca ggcctggcct ttttgattta tttgaaatac 600 aaaaaagggt
gcacaggaaa gactctgaaa gagaatgagt ccaagggttc tggagatgcg 660
cccacgcaag gggagctgta tgcgaatatc tgtgaaactc aaaaagggtc agaacaactc
720 caggagatac actatactac tccagtcttc aaagaggtgg cacccacaga
acaagaaggc 780 cttgaggata gaaaagatga ctacatctac tctgaactca
cctactaaag tgcgaagaaa 840 ctgactgtat cctaatataa gagactttcc
agtaagctga tgcttacgaa gaaacaggaa 900 attcacctgg cacttcagag
tttcatccta ggctggggca gaaggatcct gagttcaagg 960 ccagctggca
ctacatagca agaccctgtc ttaaaacaca aagatcaaaa aagatttaac 1020
ctggtcaccg aaagtgaatg tatttttcac atctggacac tattattcct ttggctaact
1080 tgggcatcta atcttgtcac caaagtagcc aggcttgata agggttaaaa
aatacttcgg 1140 ttttgtggcc ttcagccgtt ctcactttcc aacccaattt
taaatggaat tatcctgggg 1200 ctgcagaagt ggctcagcag ctaagagcag
acactgctct cccaaaggat gggagtttga 1260 gcccagagtc tacatcccat
aactcacagt tgccaataac attgtttccg aaggatccat 1320 cgccctcttc
tggcacctgc aggcactaca ctcatgtgca cagacacacc taaacgcata 1380
attaagtaag ttggggctgg agagatggct cagtggttaa gagcactgac tgctcttcca
1440 gaggtcctga gttcaatacc cagcaaccac atggtggctc acaaacatct
ataatgggat 1500 ccgatgccct cttctgctgt gtctgaagac agctatagtg
tacttatgta cataaaataa 1560 ataaatcttt aaaataaata aataagtcta
tgggcatacc accctgtaca tgcccaagct 1620 catctagaag taaataataa
aataaaaaag ttaatctta 1659 <210> SEQ ID NO 12 <211>
LENGTH: 268 <212> TYPE: PRT <213> ORGANISM: Rattus
norvegicus <400> SEQUENCE: 12 Met Trp Leu Gly Arg Gly Ile Leu
Glu Asp Glu Pro His Ser Val Ala 1 5 10 15 Ala Gln Leu Thr Met Gly
Asp Asp Asp Thr Pro Val Cys Leu Ser Val 20 25 30 Ala Ser Cys Lys
Gly Val Ser Cys Trp Leu Asp Lys Leu Leu Leu Trp 35 40 45 Ala Leu
Thr Leu Ser Ile Thr Leu Arg Asn Thr Ala Val Asp Cys Arg 50 55 60
Arg Val Asp Arg Asn Gly Leu Leu Ser Pro Asn Leu Asn Ser Ser Met 65
70 75 80 Ser Val Val Arg Met Gly Gln Asn Val Ser Leu Ser Cys Ser
Ser Lys 85 90 95 Asn Thr Ser Ile Asp Ile Thr Tyr Ser Leu Phe Leu
Gly Lys Arg Tyr 100 105 110 Leu Glu Ser Lys Arg Arg Arg Gly Gly Ala
Val Asp Phe His Leu Arg 115 120 125 Ile Ser Asn Ala Asn Glu Ser Gly
Pro Tyr Lys Cys Lys Val Asn Asp 130 135 140 Ser Asn Ser Ser Lys Tyr
Ser Gln Asn Phe Asn Phe Thr Ile Ile Gln 145 150 155 160 Asp Glu Ser
Cys Ser Ser Cys Leu Leu Ser Leu Leu Leu Pro Gly Val 165 170 175 Leu
Leu Gly Leu Ile Leu Pro Gly Leu Ala Phe Leu Ile Tyr Leu Lys 180 185
190 Tyr Lys Lys Gly Cys Thr Gly Lys Thr Leu Lys Glu Asn Glu Ser Lys
195 200 205 Gly Ser Gly Asp Ala Pro Thr Gln Gly Glu Leu Tyr Ala Asn
Ile Cys 210 215 220 Glu Thr Gln Lys Gly Ser Glu Gln Leu Gln Glu Ile
His Tyr Thr Thr 225 230 235 240 Pro Val Phe Lys Glu Val Ala Pro Thr
Glu Gln Glu Gly Leu Glu Asp 245 250 255 Arg Lys Asp Asp Tyr Ile Tyr
Ser Glu Leu Thr Tyr 260 265 <210> SEQ ID NO 13 <211>
LENGTH: 2165 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (2)...(1123) <400> SEQUENCE: 13 c atg gct gcg ccc
gca cta ggg ctg gtg tgt gga cgt tgc cct gag ctg 49 Met Ala Ala Pro
Ala Leu Gly Leu Val Cys Gly Arg Cys Pro Glu Leu 1 5 10 15 ggt ctc
gtc ctc ttg ctg ctg ctg ctc tcg ctg ctg tgt gga gcg gca 97 Gly Leu
Val Leu Leu Leu Leu Leu Leu Ser Leu Leu Cys Gly Ala Ala 20 25 30
ggg agc cag gag gcc ggg acc ggt gcg ggc gcg ggg tcc ctt gcg ggt 145
Gly Ser Gln Glu Ala Gly Thr Gly Ala Gly Ala Gly Ser Leu Ala Gly 35
40 45 tct tgc ggc tgc ggc acg ccc cag cgg cct ggc gcc cat ggc aat
tcg 193 Ser Cys Gly Cys Gly Thr Pro Gln Arg Pro Gly Ala His Gly Asn
Ser 50 55 60 gca gcc gct cac cga tac tcg cgg gag gct aac gct ccg
ggc ccc gta 241 Ala Ala Ala His Arg Tyr Ser Arg Glu Ala Asn Ala Pro
Gly Pro Val 65 70 75 80 ccc gga gag cgg caa ctc gcg cac tca aag atg
gtc ccc atc cct gtt 289 Pro Gly Glu Arg Gln Leu Ala His Ser Lys Met
Val Pro Ile Pro Val 85 90 95 gga gta ttt aca atg ggc aca gat gat
cct cag ata aag cag gat ggg 337 Gly Val Phe Thr Met Gly Thr Asp Asp
Pro Gln Ile Lys Gln Asp Gly 100 105 110 gaa gca cct gcg agg aga gtt
act att gat gcc ttt tac atg gat gcc 385 Glu Ala Pro Ala Arg Arg Val
Thr Ile Asp Ala Phe Tyr Met Asp Ala 115 120 125 tat gaa gtc agt aat
act gaa ttt gag aag ttt gtg aac tca act ggc 433 Tyr Glu Val Ser Asn
Thr Glu Phe Glu Lys Phe Val Asn Ser Thr Gly 130 135 140 tat ttg aca
gag gct gag aag ttt ggc gac tcc ttt gtc ttt gaa ggc 481 Tyr Leu Thr
Glu Ala Glu Lys Phe Gly Asp Ser Phe Val Phe Glu Gly 145 150 155 160
atg ttg agt gag caa gtg aag acc aat att caa cag gca gtt gca gct 529
Met Leu Ser Glu Gln Val Lys Thr Asn Ile Gln Gln Ala Val Ala Ala 165
170 175 gct ccc tgg tgg tta cct gtg aaa ggc gct aac tgg aga cac cca
gaa 577 Ala Pro Trp Trp Leu Pro Val Lys Gly Ala Asn Trp Arg His Pro
Glu 180 185 190 ggg cct gac tct act att ctg cac agg ccg gat cat cca
gtt ctc cat 625 Gly Pro Asp Ser Thr Ile Leu His Arg Pro Asp His Pro
Val Leu His 195 200 205 gtg tcc tgg aat gat gcg gtt gcc tac tgc act
tgg gca ggg aag cgg 673 Val Ser Trp Asn Asp Ala Val Ala Tyr Cys Thr
Trp Ala Gly Lys Arg 210 215 220 ctg ccc acg gaa gct gag tgg gaa tac
agc tgt cga gga ggc ctg cat 721 Leu Pro Thr Glu Ala Glu Trp Glu Tyr
Ser Cys Arg Gly Gly Leu His 225 230 235 240 aat aga ctt ttc ccc tgg
ggc aac aaa ctg cag ccc aaa ggc cag cat 769 Asn Arg Leu Phe Pro Trp
Gly Asn Lys Leu Gln Pro Lys Gly Gln His 245 250 255 tat gcc aac att
tgg cag ggc gag ttt ccg gtg acc aac act ggt gag 817 Tyr Ala Asn Ile
Trp Gln Gly Glu Phe Pro Val Thr Asn Thr Gly Glu 260 265 270 gat ggc
ttc caa gga act gcg cct gtt gat gcc ttc cct ccc aat ggt 865 Asp Gly
Phe Gln Gly Thr Ala Pro Val Asp Ala Phe Pro Pro Asn Gly 275 280 285
tat ggc tta tac aac ata gtg ggg aac gca tgg gaa tgg act tca gac 913
Tyr Gly Leu Tyr Asn Ile Val Gly Asn Ala Trp Glu Trp Thr Ser Asp
290 295 300 tgg tgg act gtt cat cat tct gtt gaa gaa acg ctt aac cca
aaa ggt 961 Trp Trp Thr Val His His Ser Val Glu Glu Thr Leu Asn Pro
Lys Gly 305 310 315 320 ccc cct tct ggg aaa gac cga gtg aag aaa ggt
gga tcc tac atg tgc 1009 Pro Pro Ser Gly Lys Asp Arg Val Lys Lys
Gly Gly Ser Tyr Met Cys 325 330 335 cat agg tct tat tgt tac agg tat
cgc tgt gct gct cgg agc cag aac 1057 His Arg Ser Tyr Cys Tyr Arg
Tyr Arg Cys Ala Ala Arg Ser Gln Asn 340 345 350 aca cct gat agc tct
gct tcg aat ctg gga ttc cgc tgt gca gcc gac 1105 Thr Pro Asp Ser
Ser Ala Ser Asn Leu Gly Phe Arg Cys Ala Ala Asp 355 360 365 cgc ctg
ccc act atg gac tgacaaccaa ggaaagtctt ccccagtcca 1153 Arg Leu Pro
Thr Met Asp 370 aggagcagtc gtgtctgacc tacattgggc ttttctcaga
actttgaacg atcccatgca 1213 aagaattccc accctgaggt gggttacata
cctgcccaat ggccaaagga accgccttgt 1273 gagaccaaat tgctgacctg
ggtcagtgca tgtgctttat ggtgtggtgc atctttggag 1333 atcatcgcca
tattttactt ttgagagtct ttaaagagga aggggagtgg agggaaccct 1393
gagctaggct tcaggaggcc cgcgtcctac gcaggctctg ccacaggggt tagaccccag
1453 gtccgacgct tgaccttcct gggcctcaag tgccctcccc tatcaaatga
agggatggac 1513 agcatgacct ctgggtgtct ctccaactca ccagttctaa
aaagggtatc agattctatt 1573 gtgacttcat agtgagaatt tatgatagat
tattttttag ctattttttc catgtgtgaa 1633 ccttgagtga tactaatcat
gtaaagtaag agttctctta tgtattattt tcggaagagg 1693 ggtgtggtga
ctcctttata ttcgtactgc actttgtttt tccaaggaaa tcagtgtctt 1753
ttacgttgtt atgatgaatc ccacatgggg ccggtgatgg tatgctgcag ttcagccgtt
1813 gaacacatag gaatgtctgt ggggtgactc tactgtgctt tatcttttaa
cattaagtgc 1873 ctttggttca gaggggcagt cataagctct gtttccccct
ctccccaaag ccttcagcga 1933 acgtgaaatg tgcgctaaac ggggaaacct
gtttaattct agatataggg aaaaaggaac 1993 gaggaccttg aatgagctat
attcagggta tccggtattt tgtaataggg aataggaaac 2053 cttgttggct
gtggaatatc cgatgctttg aatcatgcac tgtgttgaat aaacgtatct 2113
gctaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 2165
<210> SEQ ID NO 14 <211> LENGTH: 1122 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 14
atggctgcgc ccgcactagg gctggtgtgt ggacgttgcc ctgagctggg tctcgtcctc
60 ttgctgctgc tgctctcgct gctgtgtgga gcggcaggga gccaggaggc
cgggaccggt 120 gcgggcgcgg ggtcccttgc gggttcttgc ggctgcggca
cgccccagcg gcctggcgcc 180 catggcaatt cggcagccgc tcaccgatac
tcgcgggagg ctaacgctcc gggccccgta 240 cccggagagc ggcaactcgc
gcactcaaag atggtcccca tccctgttgg agtatttaca 300 atgggcacag
atgatcctca gataaagcag gatggggaag cacctgcgag gagagttact 360
attgatgcct tttacatgga tgcctatgaa gtcagtaata ctgaatttga gaagtttgtg
420 aactcaactg gctatttgac agaggctgag aagtttggcg actcctttgt
ctttgaaggc 480 atgttgagtg agcaagtgaa gaccaatatt caacaggcag
ttgcagctgc tccctggtgg 540 ttacctgtga aaggcgctaa ctggagacac
ccagaagggc ctgactctac tattctgcac 600 aggccggatc atccagttct
ccatgtgtcc tggaatgatg cggttgccta ctgcacttgg 660 gcagggaagc
ggctgcccac ggaagctgag tgggaataca gctgtcgagg aggcctgcat 720
aatagacttt tcccctgggg caacaaactg cagcccaaag gccagcatta tgccaacatt
780 tggcagggcg agtttccggt gaccaacact ggtgaggatg gcttccaagg
aactgcgcct 840 gttgatgcct tccctcccaa tggttatggc ttatacaaca
tagtggggaa cgcatgggaa 900 tggacttcag actggtggac tgttcatcat
tctgttgaag aaacgcttaa cccaaaaggt 960 cccccttctg ggaaagaccg
agtgaagaaa ggtggatcct acatgtgcca taggtcttat 1020 tgttacaggt
atcgctgtgc tgctcggagc cagaacacac ctgatagctc tgcttcgaat 1080
ctgggattcc gctgtgcagc cgaccgcctg cccactatgg ac 1122 <210> SEQ
ID NO 15 <211> LENGTH: 374 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 15 Met Ala Ala Pro Ala
Leu Gly Leu Val Cys Gly Arg Cys Pro Glu Leu 1 5 10 15 Gly Leu Val
Leu Leu Leu Leu Leu Leu Ser Leu Leu Cys Gly Ala Ala 20 25 30 Gly
Ser Gln Glu Ala Gly Thr Gly Ala Gly Ala Gly Ser Leu Ala Gly 35 40
45 Ser Cys Gly Cys Gly Thr Pro Gln Arg Pro Gly Ala His Gly Asn Ser
50 55 60 Ala Ala Ala His Arg Tyr Ser Arg Glu Ala Asn Ala Pro Gly
Pro Val 65 70 75 80 Pro Gly Glu Arg Gln Leu Ala His Ser Lys Met Val
Pro Ile Pro Val 85 90 95 Gly Val Phe Thr Met Gly Thr Asp Asp Pro
Gln Ile Lys Gln Asp Gly 100 105 110 Glu Ala Pro Ala Arg Arg Val Thr
Ile Asp Ala Phe Tyr Met Asp Ala 115 120 125 Tyr Glu Val Ser Asn Thr
Glu Phe Glu Lys Phe Val Asn Ser Thr Gly 130 135 140 Tyr Leu Thr Glu
Ala Glu Lys Phe Gly Asp Ser Phe Val Phe Glu Gly 145 150 155 160 Met
Leu Ser Glu Gln Val Lys Thr Asn Ile Gln Gln Ala Val Ala Ala 165 170
175 Ala Pro Trp Trp Leu Pro Val Lys Gly Ala Asn Trp Arg His Pro Glu
180 185 190 Gly Pro Asp Ser Thr Ile Leu His Arg Pro Asp His Pro Val
Leu His 195 200 205 Val Ser Trp Asn Asp Ala Val Ala Tyr Cys Thr Trp
Ala Gly Lys Arg 210 215 220 Leu Pro Thr Glu Ala Glu Trp Glu Tyr Ser
Cys Arg Gly Gly Leu His 225 230 235 240 Asn Arg Leu Phe Pro Trp Gly
Asn Lys Leu Gln Pro Lys Gly Gln His 245 250 255 Tyr Ala Asn Ile Trp
Gln Gly Glu Phe Pro Val Thr Asn Thr Gly Glu 260 265 270 Asp Gly Phe
Gln Gly Thr Ala Pro Val Asp Ala Phe Pro Pro Asn Gly 275 280 285 Tyr
Gly Leu Tyr Asn Ile Val Gly Asn Ala Trp Glu Trp Thr Ser Asp 290 295
300 Trp Trp Thr Val His His Ser Val Glu Glu Thr Leu Asn Pro Lys Gly
305 310 315 320 Pro Pro Ser Gly Lys Asp Arg Val Lys Lys Gly Gly Ser
Tyr Met Cys 325 330 335 His Arg Ser Tyr Cys Tyr Arg Tyr Arg Cys Ala
Ala Arg Ser Gln Asn 340 345 350 Thr Pro Asp Ser Ser Ala Ser Asn Leu
Gly Phe Arg Cys Ala Ala Asp 355 360 365 Arg Leu Pro Thr Met Asp 370
<210> SEQ ID NO 16 <211> LENGTH: 340 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 16 Gln
Glu Ala Gly Thr Gly Ala Gly Ala Gly Ser Leu Ala Gly Ser Cys 1 5 10
15 Gly Cys Gly Thr Pro Gln Arg Pro Gly Ala His Gly Asn Ser Ala Ala
20 25 30 Ala His Arg Tyr Ser Arg Glu Ala Asn Ala Pro Gly Pro Val
Pro Gly 35 40 45 Glu Arg Gln Leu Ala His Ser Lys Met Val Pro Ile
Pro Val Gly Val 50 55 60 Phe Thr Met Gly Thr Asp Asp Pro Gln Ile
Lys Gln Asp Gly Glu Ala 65 70 75 80 Pro Ala Arg Arg Val Thr Ile Asp
Ala Phe Tyr Met Asp Ala Tyr Glu 85 90 95 Val Ser Asn Thr Glu Phe
Glu Lys Phe Val Asn Ser Thr Gly Tyr Leu 100 105 110 Thr Glu Ala Glu
Lys Phe Gly Asp Ser Phe Val Phe Glu Gly Met Leu 115 120 125 Ser Glu
Gln Val Lys Thr Asn Ile Gln Gln Ala Val Ala Ala Ala Pro 130 135 140
Trp Trp Leu Pro Val Lys Gly Ala Asn Trp Arg His Pro Glu Gly Pro 145
150 155 160 Asp Ser Thr Ile Leu His Arg Pro Asp His Pro Val Leu His
Val Ser 165 170 175 Trp Asn Asp Ala Val Ala Tyr Cys Thr Trp Ala Gly
Lys Arg Leu Pro 180 185 190 Thr Glu Ala Glu Trp Glu Tyr Ser Cys Arg
Gly Gly Leu His Asn Arg 195 200 205 Leu Phe Pro Trp Gly Asn Lys Leu
Gln Pro Lys Gly Gln His Tyr Ala 210 215 220 Asn Ile Trp Gln Gly Glu
Phe Pro Val Thr Asn Thr Gly Glu Asp Gly 225 230 235 240 Phe Gln Gly
Thr Ala Pro Val Asp Ala Phe Pro Pro Asn Gly Tyr Gly 245 250 255 Leu
Tyr Asn Ile Val Gly Asn Ala Trp Glu Trp Thr Ser Asp Trp Trp 260 265
270 Thr Val His His Ser Val Glu Glu Thr Leu Asn Pro Lys Gly Pro Pro
275 280 285 Ser Gly Lys Asp Arg Val Lys Lys Gly Gly Ser Tyr Met Cys
His Arg 290 295 300 Ser Tyr Cys Tyr Arg Tyr Arg Cys Ala Ala Arg Ser
Gln Asn Thr Pro 305 310 315 320 Asp Ser Ser Ala Ser Asn Leu Gly Phe
Arg Cys Ala Ala Asp Arg Leu
325 330 335 Pro Thr Met Asp 340 <210> SEQ ID NO 17
<211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY: SIGNAL
<222> LOCATION: (1)...(34) <400> SEQUENCE: 17 Met Ala
Ala Pro Ala Leu Gly Leu Val Cys Gly Arg Cys Pro Glu Leu -30 -25 -20
Gly Leu Val Leu Leu Leu Leu Leu Leu Ser Leu Leu Cys Gly Ala Ala -15
-10 -5 Gly Ser <210> SEQ ID NO 18 <211> LENGTH: 299
<212> TYPE: PRT <213> ORGANISM: Mycobacterium
tuberculosis <400> SEQUENCE: 18 Met Leu Thr Glu Leu Val Asp
Leu Pro Gly Gly Ser Phe Arg Met Gly 1 5 10 15 Ser Thr Arg Phe Tyr
Pro Glu Glu Ala Pro Ile His Thr Val Thr Val 20 25 30 Arg Ala Phe
Ala Val Glu Arg His Pro Val Thr Asn Ala Gln Phe Ala 35 40 45 Glu
Phe Val Ser Ala Thr Gly Tyr Val Thr Val Ala Glu Gln Pro Leu 50 55
60 Asp Pro Gly Leu Tyr Pro Gly Val Asp Ala Ala Asp Leu Cys Pro Gly
65 70 75 80 Ala Met Val Phe Cys Pro Thr Ala Gly Pro Val Asp Leu Arg
Asp Trp 85 90 95 Arg Gln Trp Trp Asp Trp Val Pro Gly Ala Cys Trp
Arg His Pro Phe 100 105 110 Gly Arg Asp Ser Asp Ile Ala Asp Arg Ala
Gly His Pro Val Val Gln 115 120 125 Val Ala Tyr Pro Asp Ala Val Ala
Tyr Ala Arg Trp Ala Gly Arg Arg 130 135 140 Leu Pro Thr Glu Ala Glu
Trp Glu Tyr Ala Ala Arg Gly Gly Thr Thr 145 150 155 160 Ala Thr Tyr
Ala Trp Gly Asp Gln Glu Lys Pro Gly Gly Met Leu Met 165 170 175 Ala
Asn Thr Trp Gln Gly Arg Phe Pro Tyr Arg Asn Asp Gly Ala Leu 180 185
190 Gly Trp Val Gly Thr Ser Pro Val Gly Arg Phe Pro Ala Asn Gly Phe
195 200 205 Gly Leu Leu Asp Met Ile Gly Asn Val Trp Glu Trp Thr Thr
Thr Glu 210 215 220 Phe Tyr Pro His His Arg Ile Asp Pro Pro Ser Thr
Ala Cys Cys Ala 225 230 235 240 Pro Val Lys Leu Ala Thr Ala Ala Asp
Pro Thr Ile Ser Gln Thr Leu 245 250 255 Lys Gly Gly Ser His Leu Cys
Ala Pro Glu Tyr Cys His Arg Tyr Arg 260 265 270 Pro Ala Ala Arg Ser
Pro Gln Ser Gln Asp Thr Ala Thr Thr His Ile 275 280 285 Gly Phe Arg
Cys Val Ala Asp Pro Val Ser Gly 290 295 <210> SEQ ID NO 19
<211> LENGTH: 2811 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (183)...(2567) <400> SEQUENCE: 19
ctcggcccgc tcggcgcgcc ccttcccagc cgcccttccg taccggctct cgggctcttc
60 cggtctccgg ccgcccctta cctgcaggct cttctcccgc cgcggcccgg
cgctctccga 120 gtcgcccctg cggactggtc tcgcacagtg cctgggcacc
gggcgccaga cagacactgg 180 cc atg acg agc ggc gca acc agg tac cgg
ctg agc tgc tcg ctc cgg 227 Met Thr Ser Gly Ala Thr Arg Tyr Arg Leu
Ser Cys Ser Leu Arg 1 5 10 15 ggc cac gag ctg gac gta cgg ggc ctg
gtg tgc tgc gcc tat ccg ccg 275 Gly His Glu Leu Asp Val Arg Gly Leu
Val Cys Cys Ala Tyr Pro Pro 20 25 30 gga gcc ttt gtg tcc gtg tcc
cga gac cgc acc acc cgc ctc tgg gcc 323 Gly Ala Phe Val Ser Val Ser
Arg Asp Arg Thr Thr Arg Leu Trp Ala 35 40 45 cca gac agt cca aac
agg agc ttt aca gaa atg cac tgt atg agt ggc 371 Pro Asp Ser Pro Asn
Arg Ser Phe Thr Glu Met His Cys Met Ser Gly 50 55 60 cat tcc aat
ttt gta tct tgt gta tgc atc ata ccc tca agt gac atc 419 His Ser Asn
Phe Val Ser Cys Val Cys Ile Ile Pro Ser Ser Asp Ile 65 70 75 tac
cct cat ggc cta att gcc acc ggt gga aat gac cac aat ata tgc 467 Tyr
Pro His Gly Leu Ile Ala Thr Gly Gly Asn Asp His Asn Ile Cys 80 85
90 95 att ttc tca ctg gac agt cca atg cca ctt tat att cta aaa ggc
cac 515 Ile Phe Ser Leu Asp Ser Pro Met Pro Leu Tyr Ile Leu Lys Gly
His 100 105 110 aaa aat act gtt tgt agt cta tca tct gga aaa ttt ggg
aca tta ctt 563 Lys Asn Thr Val Cys Ser Leu Ser Ser Gly Lys Phe Gly
Thr Leu Leu 115 120 125 agt ggt tca tgg gac acc act gct aaa gtc tgg
ctg aat gac aag tgc 611 Ser Gly Ser Trp Asp Thr Thr Ala Lys Val Trp
Leu Asn Asp Lys Cys 130 135 140 atg atg acc ttg cag ggt cat aca gct
gca gtg tgg gcg gta aag atc 659 Met Met Thr Leu Gln Gly His Thr Ala
Ala Val Trp Ala Val Lys Ile 145 150 155 tta cct gaa cag ggc tta atg
ttg act gga tca gca gac aag act gtt 707 Leu Pro Glu Gln Gly Leu Met
Leu Thr Gly Ser Ala Asp Lys Thr Val 160 165 170 175 aaa ctg tgg aag
gct gga aga tgt gag agg act ttt tca ggg cat gaa 755 Lys Leu Trp Lys
Ala Gly Arg Cys Glu Arg Thr Phe Ser Gly His Glu 180 185 190 gac tgt
gta aga ggt ttg gca att ttg agt gaa aca gaa ttt ctt tcc 803 Asp Cys
Val Arg Gly Leu Ala Ile Leu Ser Glu Thr Glu Phe Leu Ser 195 200 205
tgt gca aat gat gct agt att aga agg tgg caa atc act ggc gag tgt 851
Cys Ala Asn Asp Ala Ser Ile Arg Arg Trp Gln Ile Thr Gly Glu Cys 210
215 220 ctt gaa gta tat tat gga cat aca aat tat att tat agc ata tcc
gtt 899 Leu Glu Val Tyr Tyr Gly His Thr Asn Tyr Ile Tyr Ser Ile Ser
Val 225 230 235 ttt cca aat tgt aga gac ttt gtg aca aca gca gag gac
aga tct ctg 947 Phe Pro Asn Cys Arg Asp Phe Val Thr Thr Ala Glu Asp
Arg Ser Leu 240 245 250 255 aga atc tgg aaa cat ggg gaa tgt gct caa
act atc cga ctt cca gct 995 Arg Ile Trp Lys His Gly Glu Cys Ala Gln
Thr Ile Arg Leu Pro Ala 260 265 270 cag tct ata tgg tgc tgc tgt gtg
ctc gac aat ggt gac att gtg gtt 1043 Gln Ser Ile Trp Cys Cys Cys
Val Leu Asp Asn Gly Asp Ile Val Val 275 280 285 ggt gcg agt gat ggc
att att aga gtg ttt aca gaa tca gaa gat cga 1091 Gly Ala Ser Asp
Gly Ile Ile Arg Val Phe Thr Glu Ser Glu Asp Arg 290 295 300 aca gca
agt gct gaa gaa atc aag gct ttt gaa aaa gaa ctg tct cac 1139 Thr
Ala Ser Ala Glu Glu Ile Lys Ala Phe Glu Lys Glu Leu Ser His 305 310
315 gca acc att gat tct aaa act ggc gat tta ggg gac atc aat gct gag
1187 Ala Thr Ile Asp Ser Lys Thr Gly Asp Leu Gly Asp Ile Asn Ala
Glu 320 325 330 335 cag ctt cct ggg agg gaa cat ctt aat gaa cct ggt
act aga gaa gga 1235 Gln Leu Pro Gly Arg Glu His Leu Asn Glu Pro
Gly Thr Arg Glu Gly 340 345 350 cag act cgt cta atc aga gat ggg gag
aaa gtc gaa gcc tat cag tgg 1283 Gln Thr Arg Leu Ile Arg Asp Gly
Glu Lys Val Glu Ala Tyr Gln Trp 355 360 365 agt gtt agt gaa ggg agg
tgg ata aaa att ggt gat gtt gtt ggc tca 1331 Ser Val Ser Glu Gly
Arg Trp Ile Lys Ile Gly Asp Val Val Gly Ser 370 375 380 tct ggt gct
aat cag caa aca tct gga aaa gtt tta tat gaa ggg aaa 1379 Ser Gly
Ala Asn Gln Gln Thr Ser Gly Lys Val Leu Tyr Glu Gly Lys 385 390 395
gaa ttt gat tat gtt ttc tca att gat gtc aat gaa ggt gga cca tca
1427 Glu Phe Asp Tyr Val Phe Ser Ile Asp Val Asn Glu Gly Gly Pro
Ser 400 405 410 415 tat aaa ttg cca tat aat acc agt gat gac cct tgg
tta act gca tac 1475 Tyr Lys Leu Pro Tyr Asn Thr Ser Asp Asp Pro
Trp Leu Thr Ala Tyr 420 425 430 aac ttc tta cag aag aat gat ttg aat
cct atg ttt ctg gat caa gta 1523 Asn Phe Leu Gln Lys Asn Asp Leu
Asn Pro Met Phe Leu Asp Gln Val 435 440 445 gct aaa ttt att att gat
aac aca aaa ggt caa atg ttg gga ctt ggg 1571 Ala Lys Phe Ile Ile
Asp Asn Thr Lys Gly Gln Met Leu Gly Leu Gly 450 455 460 aat ccc agc
ttt tca gat cca ttt aca ggt ggt ggt cgg tat gtt ccg 1619 Asn Pro
Ser Phe Ser Asp Pro Phe Thr Gly Gly Gly Arg Tyr Val Pro 465 470 475
ggc tct tcg gga tct tct aac aca cta ccc aca gca gat cct ttt aca
1667 Gly Ser Ser Gly Ser Ser Asn Thr Leu Pro Thr Ala Asp Pro Phe
Thr 480 485 490 495 ggt gct ggt cgt tat gta cca ggt tct gca agt atg
gga act acc atg 1715 Gly Ala Gly Arg Tyr Val Pro Gly Ser Ala Ser
Met Gly Thr Thr Met 500 505 510 gcc gga gtt gat cca ttt aca ggg aat
agt gcc tac cga tca gct gca 1763 Ala Gly Val Asp Pro Phe Thr Gly
Asn Ser Ala Tyr Arg Ser Ala Ala 515 520 525 tct aaa aca atg aat att
tat ttc cct aaa aaa gag gct gtc aca ttt 1811 Ser Lys Thr Met Asn
Ile Tyr Phe Pro Lys Lys Glu Ala Val Thr Phe 530 535 540 gac caa gca
aac cct aca caa ata tta ggt aaa ctg aag gaa ctt aat 1859 Asp Gln
Ala Asn Pro Thr Gln Ile Leu Gly Lys Leu Lys Glu Leu Asn 545 550 555
gga act gca cct gaa gag aag aag tta act gag gat gac ttg ata ctt
1907 Gly Thr Ala Pro Glu Glu Lys Lys Leu Thr Glu Asp Asp Leu Ile
Leu 560 565 570 575 ctt gag aag ata ctg tct cta ata tgt aat agt tct
tca gaa aaa ccc 1955
Leu Glu Lys Ile Leu Ser Leu Ile Cys Asn Ser Ser Ser Glu Lys Pro 580
585 590 aca gtc cag caa ctt cag att ttg tgg aaa gct att aac tgt cct
gaa 2003 Thr Val Gln Gln Leu Gln Ile Leu Trp Lys Ala Ile Asn Cys
Pro Glu 595 600 605 gat att gtc ttt cct gca ctt gac att ctt cgg ttg
tca att aaa cac 2051 Asp Ile Val Phe Pro Ala Leu Asp Ile Leu Arg
Leu Ser Ile Lys His 610 615 620 ccc agt gtg aat gag aac ttc tgc aat
gaa aag gaa ggg gct cag ttc 2099 Pro Ser Val Asn Glu Asn Phe Cys
Asn Glu Lys Glu Gly Ala Gln Phe 625 630 635 agc agt cat ctt atc aat
ctt ctg aac cct aaa gga aag cca gca aac 2147 Ser Ser His Leu Ile
Asn Leu Leu Asn Pro Lys Gly Lys Pro Ala Asn 640 645 650 655 cag ctg
ctt gct ctc agg act ttt tgc aat tgt ttt gtt ggc cag gca 2195 Gln
Leu Leu Ala Leu Arg Thr Phe Cys Asn Cys Phe Val Gly Gln Ala 660 665
670 gga caa aaa ctc atg atg tcc cag agg gaa tca ctg atg tcc cat gca
2243 Gly Gln Lys Leu Met Met Ser Gln Arg Glu Ser Leu Met Ser His
Ala 675 680 685 ata gaa ctg aaa tca ggg agc aat aag aac att cac att
gct ctg gct 2291 Ile Glu Leu Lys Ser Gly Ser Asn Lys Asn Ile His
Ile Ala Leu Ala 690 695 700 aca ttg gcc ctg aac tat tct gtt tgt ttt
cat aaa gac cat aac att 2339 Thr Leu Ala Leu Asn Tyr Ser Val Cys
Phe His Lys Asp His Asn Ile 705 710 715 gaa ggg aaa gcc caa tgt ttg
tca cta att agc aca atc ttg gaa gta 2387 Glu Gly Lys Ala Gln Cys
Leu Ser Leu Ile Ser Thr Ile Leu Glu Val 720 725 730 735 gta caa gac
cta gaa gcc act ttt aga ctt ctt gtg gct ctt gga aca 2435 Val Gln
Asp Leu Glu Ala Thr Phe Arg Leu Leu Val Ala Leu Gly Thr 740 745 750
ctt atc agt gat gat tca aat gct gta caa tta gcc aag tct tta ggt
2483 Leu Ile Ser Asp Asp Ser Asn Ala Val Gln Leu Ala Lys Ser Leu
Gly 755 760 765 gtt gat tct caa ata aaa aag tat tcc tca gta tca gaa
cca gct aaa 2531 Val Asp Ser Gln Ile Lys Lys Tyr Ser Ser Val Ser
Glu Pro Ala Lys 770 775 780 gta agt gaa tgc tgt aga ttt atc cta aat
ttg ctg tagcagtggg 2577 Val Ser Glu Cys Cys Arg Phe Ile Leu Asn Leu
Leu 785 790 795 gaagagggac ggatattttt aattgattag tgtttttttc
ctcacatttg acatgactga 2637 taacagataa ttaaaaaaag agaatacggt
ggattaagta aaattttaca tcttgtaaag 2697 tggtggggag gggaaacaga
aataaaattt ttgcactgct gaaaaaaaaa aaaaaaaaaa 2757 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 2811 <210>
SEQ ID NO 20 <211> LENGTH: 2385 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 20
atgacgagcg gcgcaaccag gtaccggctg agctgctcgc tccggggcca cgagctggac
60 gtacggggcc tggtgtgctg cgcctatccg ccgggagcct ttgtgtccgt
gtcccgagac 120 cgcaccaccc gcctctgggc cccagacagt ccaaacagga
gctttacaga aatgcactgt 180 atgagtggcc attccaattt tgtatcttgt
gtatgcatca taccctcaag tgacatctac 240 cctcatggcc taattgccac
cggtggaaat gaccacaata tatgcatttt ctcactggac 300 agtccaatgc
cactttatat tctaaaaggc cacaaaaata ctgtttgtag tctatcatct 360
ggaaaatttg ggacattact tagtggttca tgggacacca ctgctaaagt ctggctgaat
420 gacaagtgca tgatgacctt gcagggtcat acagctgcag tgtgggcggt
aaagatctta 480 cctgaacagg gcttaatgtt gactggatca gcagacaaga
ctgttaaact gtggaaggct 540 ggaagatgtg agaggacttt ttcagggcat
gaagactgtg taagaggttt ggcaattttg 600 agtgaaacag aatttctttc
ctgtgcaaat gatgctagta ttagaaggtg gcaaatcact 660 ggcgagtgtc
ttgaagtata ttatggacat acaaattata tttatagcat atccgttttt 720
ccaaattgta gagactttgt gacaacagca gaggacagat ctctgagaat ctggaaacat
780 ggggaatgtg ctcaaactat ccgacttcca gctcagtcta tatggtgctg
ctgtgtgctc 840 gacaatggtg acattgtggt tggtgcgagt gatggcatta
ttagagtgtt tacagaatca 900 gaagatcgaa cagcaagtgc tgaagaaatc
aaggcttttg aaaaagaact gtctcacgca 960 accattgatt ctaaaactgg
cgatttaggg gacatcaatg ctgagcagct tcctgggagg 1020 gaacatctta
atgaacctgg tactagagaa ggacagactc gtctaatcag agatggggag 1080
aaagtcgaag cctatcagtg gagtgttagt gaagggaggt ggataaaaat tggtgatgtt
1140 gttggctcat ctggtgctaa tcagcaaaca tctggaaaag ttttatatga
agggaaagaa 1200 tttgattatg ttttctcaat tgatgtcaat gaaggtggac
catcatataa attgccatat 1260 aataccagtg atgacccttg gttaactgca
tacaacttct tacagaagaa tgatttgaat 1320 cctatgtttc tggatcaagt
agctaaattt attattgata acacaaaagg tcaaatgttg 1380 ggacttggga
atcccagctt ttcagatcca tttacaggtg gtggtcggta tgttccgggc 1440
tcttcgggat cttctaacac actacccaca gcagatcctt ttacaggtgc tggtcgttat
1500 gtaccaggtt ctgcaagtat gggaactacc atggccggag ttgatccatt
tacagggaat 1560 agtgcctacc gatcagctgc atctaaaaca atgaatattt
atttccctaa aaaagaggct 1620 gtcacatttg accaagcaaa ccctacacaa
atattaggta aactgaagga acttaatgga 1680 actgcacctg aagagaagaa
gttaactgag gatgacttga tacttcttga gaagatactg 1740 tctctaatat
gtaatagttc ttcagaaaaa cccacagtcc agcaacttca gattttgtgg 1800
aaagctatta actgtcctga agatattgtc tttcctgcac ttgacattct tcggttgtca
1860 attaaacacc ccagtgtgaa tgagaacttc tgcaatgaaa aggaaggggc
tcagttcagc 1920 agtcatctta tcaatcttct gaaccctaaa ggaaagccag
caaaccagct gcttgctctc 1980 aggacttttt gcaattgttt tgttggccag
gcaggacaaa aactcatgat gtcccagagg 2040 gaatcactga tgtcccatgc
aatagaactg aaatcaggga gcaataagaa cattcacatt 2100 gctctggcta
cattggccct gaactattct gtttgttttc ataaagacca taacattgaa 2160
gggaaagccc aatgtttgtc actaattagc acaatcttgg aagtagtaca agacctagaa
2220 gccactttta gacttcttgt ggctcttgga acacttatca gtgatgattc
aaatgctgta 2280 caattagcca agtctttagg tgttgattct caaataaaaa
agtattcctc agtatcagaa 2340 ccagctaaag taagtgaatg ctgtagattt
atcctaaatt tgctg 2385 <210> SEQ ID NO 21 <211> LENGTH:
795 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 21 Met Thr Ser Gly Ala Thr Arg Tyr Arg Leu
Ser Cys Ser Leu Arg Gly 1 5 10 15 His Glu Leu Asp Val Arg Gly Leu
Val Cys Cys Ala Tyr Pro Pro Gly 20 25 30 Ala Phe Val Ser Val Ser
Arg Asp Arg Thr Thr Arg Leu Trp Ala Pro 35 40 45 Asp Ser Pro Asn
Arg Ser Phe Thr Glu Met His Cys Met Ser Gly His 50 55 60 Ser Asn
Phe Val Ser Cys Val Cys Ile Ile Pro Ser Ser Asp Ile Tyr 65 70 75 80
Pro His Gly Leu Ile Ala Thr Gly Gly Asn Asp His Asn Ile Cys Ile 85
90 95 Phe Ser Leu Asp Ser Pro Met Pro Leu Tyr Ile Leu Lys Gly His
Lys 100 105 110 Asn Thr Val Cys Ser Leu Ser Ser Gly Lys Phe Gly Thr
Leu Leu Ser 115 120 125 Gly Ser Trp Asp Thr Thr Ala Lys Val Trp Leu
Asn Asp Lys Cys Met 130 135 140 Met Thr Leu Gln Gly His Thr Ala Ala
Val Trp Ala Val Lys Ile Leu 145 150 155 160 Pro Glu Gln Gly Leu Met
Leu Thr Gly Ser Ala Asp Lys Thr Val Lys 165 170 175 Leu Trp Lys Ala
Gly Arg Cys Glu Arg Thr Phe Ser Gly His Glu Asp 180 185 190 Cys Val
Arg Gly Leu Ala Ile Leu Ser Glu Thr Glu Phe Leu Ser Cys 195 200 205
Ala Asn Asp Ala Ser Ile Arg Arg Trp Gln Ile Thr Gly Glu Cys Leu 210
215 220 Glu Val Tyr Tyr Gly His Thr Asn Tyr Ile Tyr Ser Ile Ser Val
Phe 225 230 235 240 Pro Asn Cys Arg Asp Phe Val Thr Thr Ala Glu Asp
Arg Ser Leu Arg 245 250 255 Ile Trp Lys His Gly Glu Cys Ala Gln Thr
Ile Arg Leu Pro Ala Gln 260 265 270 Ser Ile Trp Cys Cys Cys Val Leu
Asp Asn Gly Asp Ile Val Val Gly 275 280 285 Ala Ser Asp Gly Ile Ile
Arg Val Phe Thr Glu Ser Glu Asp Arg Thr 290 295 300 Ala Ser Ala Glu
Glu Ile Lys Ala Phe Glu Lys Glu Leu Ser His Ala 305 310 315 320 Thr
Ile Asp Ser Lys Thr Gly Asp Leu Gly Asp Ile Asn Ala Glu Gln 325 330
335 Leu Pro Gly Arg Glu His Leu Asn Glu Pro Gly Thr Arg Glu Gly Gln
340 345 350 Thr Arg Leu Ile Arg Asp Gly Glu Lys Val Glu Ala Tyr Gln
Trp Ser 355 360 365 Val Ser Glu Gly Arg Trp Ile Lys Ile Gly Asp Val
Val Gly Ser Ser 370 375 380 Gly Ala Asn Gln Gln Thr Ser Gly Lys Val
Leu Tyr Glu Gly Lys Glu 385 390 395 400 Phe Asp Tyr Val Phe Ser Ile
Asp Val Asn Glu Gly Gly Pro Ser Tyr 405 410 415 Lys Leu Pro Tyr Asn
Thr Ser Asp Asp Pro Trp Leu Thr Ala Tyr Asn 420 425 430 Phe Leu Gln
Lys Asn Asp Leu Asn Pro Met Phe Leu Asp Gln Val Ala 435 440 445 Lys
Phe Ile Ile Asp Asn Thr Lys Gly Gln Met Leu Gly Leu Gly Asn 450 455
460 Pro Ser Phe Ser Asp Pro Phe Thr Gly Gly Gly Arg Tyr Val Pro Gly
465 470 475 480 Ser Ser Gly Ser Ser Asn Thr Leu Pro Thr Ala Asp Pro
Phe Thr Gly 485 490 495
Ala Gly Arg Tyr Val Pro Gly Ser Ala Ser Met Gly Thr Thr Met Ala 500
505 510 Gly Val Asp Pro Phe Thr Gly Asn Ser Ala Tyr Arg Ser Ala Ala
Ser 515 520 525 Lys Thr Met Asn Ile Tyr Phe Pro Lys Lys Glu Ala Val
Thr Phe Asp 530 535 540 Gln Ala Asn Pro Thr Gln Ile Leu Gly Lys Leu
Lys Glu Leu Asn Gly 545 550 555 560 Thr Ala Pro Glu Glu Lys Lys Leu
Thr Glu Asp Asp Leu Ile Leu Leu 565 570 575 Glu Lys Ile Leu Ser Leu
Ile Cys Asn Ser Ser Ser Glu Lys Pro Thr 580 585 590 Val Gln Gln Leu
Gln Ile Leu Trp Lys Ala Ile Asn Cys Pro Glu Asp 595 600 605 Ile Val
Phe Pro Ala Leu Asp Ile Leu Arg Leu Ser Ile Lys His Pro 610 615 620
Ser Val Asn Glu Asn Phe Cys Asn Glu Lys Glu Gly Ala Gln Phe Ser 625
630 635 640 Ser His Leu Ile Asn Leu Leu Asn Pro Lys Gly Lys Pro Ala
Asn Gln 645 650 655 Leu Leu Ala Leu Arg Thr Phe Cys Asn Cys Phe Val
Gly Gln Ala Gly 660 665 670 Gln Lys Leu Met Met Ser Gln Arg Glu Ser
Leu Met Ser His Ala Ile 675 680 685 Glu Leu Lys Ser Gly Ser Asn Lys
Asn Ile His Ile Ala Leu Ala Thr 690 695 700 Leu Ala Leu Asn Tyr Ser
Val Cys Phe His Lys Asp His Asn Ile Glu 705 710 715 720 Gly Lys Ala
Gln Cys Leu Ser Leu Ile Ser Thr Ile Leu Glu Val Val 725 730 735 Gln
Asp Leu Glu Ala Thr Phe Arg Leu Leu Val Ala Leu Gly Thr Leu 740 745
750 Ile Ser Asp Asp Ser Asn Ala Val Gln Leu Ala Lys Ser Leu Gly Val
755 760 765 Asp Ser Gln Ile Lys Lys Tyr Ser Ser Val Ser Glu Pro Ala
Lys Val 770 775 780 Ser Glu Cys Cys Arg Phe Ile Leu Asn Leu Leu 785
790 795 <210> SEQ ID NO 22 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
22 Gly His Glu Leu Asp Val Arg Gly Leu Val Cys Cys Ala Tyr Pro Pro
1 5 10 15 Gly Ala Phe Val Ser Val Ser Arg Asp Arg Thr Thr Arg Leu
Trp 20 25 30 <210> SEQ ID NO 23 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 23 Gly His Ser Asn Phe Val Ser Cys Val Cys
Ile Ile Pro Ser Ser Asp 1 5 10 15 Ile Tyr Pro His Gly Leu Ile Ala
Thr Gly Gly Asn Asp His Asn Ile 20 25 30 Cys Ile Phe 35 <210>
SEQ ID NO 24 <211> LENGTH: 29 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 24 Gly His
Lys Asn Thr Val Cys Ser Leu Ser Ser Gly Lys Phe Gly Thr 1 5 10 15
Leu Leu Ser Gly Ser Trp Asp Thr Thr Ala Lys Val Trp 20 25
<210> SEQ ID NO 25 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 25 Gly
His Thr Ala Ala Val Trp Ala Val Lys Ile Leu Pro Glu Gln Gly 1 5 10
15 Leu Met Leu Thr Gly Ser Ala Asp Lys Thr Val Lys Leu Trp 20 25 30
<210> SEQ ID NO 26 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 26 Gly
His Glu Asp Cys Val Arg Gly Leu Ala Ile Leu Ser Glu Thr Glu 1 5 10
15 Phe Leu Ser Cys Ala Asn Asp Ala Ser Ile Arg Arg Trp 20 25
<210> SEQ ID NO 27 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 27 Gly
His Thr Asn Tyr Ile Tyr Ser Ile Ser Val Phe Pro Asn Cys Arg 1 5 10
15 Asp Phe Val Thr Thr Ala Glu Asp Arg Ser Leu Arg Ile Trp 20 25 30
<210> SEQ ID NO 28 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 28 Gly
Glu Cys Ala Gln Thr Ile Arg Leu Pro Ala Gln Ser Ile Trp Cys 1 5 10
15 Cys Cys Val Leu Asp Asn Gly Asp Ile Val Val Gly Ala Ser Asp Gly
20 25 30 Ile Ile Arg Val Phe 35 <210> SEQ ID NO 29
<211> LENGTH: 2217 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (1)...(2214) <400> SEQUENCE: 29 atg
cac tgt atg agc ggc cac tcc aat ttt gta tct tgt gta tgc atc 48 Met
His Cys Met Ser Gly His Ser Asn Phe Val Ser Cys Val Cys Ile 1 5 10
15 ata ccc tca agt gac atc tac cct cat ggc cta att gcc acc ggt gga
96 Ile Pro Ser Ser Asp Ile Tyr Pro His Gly Leu Ile Ala Thr Gly Gly
20 25 30 aat gac cac aat ata tgc att ttc tca ctg gac agt cca atg
cca ctt 144 Asn Asp His Asn Ile Cys Ile Phe Ser Leu Asp Ser Pro Met
Pro Leu 35 40 45 tat att cta aaa ggc cac aaa aat act gtt tgt agt
cta tca tct gga 192 Tyr Ile Leu Lys Gly His Lys Asn Thr Val Cys Ser
Leu Ser Ser Gly 50 55 60 aaa ttt ggg aca tta ctt agt ggt tca tgg
gac acc act gct aaa gtc 240 Lys Phe Gly Thr Leu Leu Ser Gly Ser Trp
Asp Thr Thr Ala Lys Val 65 70 75 80 tgg ctg aat gac aag tgc atg atg
acc ttg cag ggt cat aca gct gca 288 Trp Leu Asn Asp Lys Cys Met Met
Thr Leu Gln Gly His Thr Ala Ala 85 90 95 gtg tgg gcg gta aag atc
tta cct gaa cag ggc tta atg ttg act gga 336 Val Trp Ala Val Lys Ile
Leu Pro Glu Gln Gly Leu Met Leu Thr Gly 100 105 110 tca gca gac aag
act gtt aaa ctg tgg aag gct gga aga tgt gag agg 384 Ser Ala Asp Lys
Thr Val Lys Leu Trp Lys Ala Gly Arg Cys Glu Arg 115 120 125 act ttt
tca ggg cat gaa gac tgt gta aga ggt ttg gca att ttg agt 432 Thr Phe
Ser Gly His Glu Asp Cys Val Arg Gly Leu Ala Ile Leu Ser 130 135 140
gaa aca gaa ttt ctt tcc tgt gca aat gat gct agt att aga agg tgg 480
Glu Thr Glu Phe Leu Ser Cys Ala Asn Asp Ala Ser Ile Arg Arg Trp 145
150 155 160 caa atc act ggc gag tgt ctt gaa gta tat tat gga cat aca
aat tat 528 Gln Ile Thr Gly Glu Cys Leu Glu Val Tyr Tyr Gly His Thr
Asn Tyr 165 170 175 att tat agc ata tcc gtt ttt cca aat tgt aga gac
ttt gtg aca aca 576 Ile Tyr Ser Ile Ser Val Phe Pro Asn Cys Arg Asp
Phe Val Thr Thr 180 185 190 gca gag gac aga tct ctg aga atc tgg aaa
cat ggg gaa tgt gct caa 624 Ala Glu Asp Arg Ser Leu Arg Ile Trp Lys
His Gly Glu Cys Ala Gln 195 200 205 act atc cga ctt cca gct cag tct
ata tgg tgc tgc tgt gtg ctc gac 672 Thr Ile Arg Leu Pro Ala Gln Ser
Ile Trp Cys Cys Cys Val Leu Asp 210 215 220 aat ggt gac att gtg gtt
ggt gcg agt gat ggc att att aga gtg ttt 720 Asn Gly Asp Ile Val Val
Gly Ala Ser Asp Gly Ile Ile Arg Val Phe 225 230 235 240 aca gag tca
gaa gat cga aca gca agt gct gaa gaa atc aag gct ttt 768 Thr Glu Ser
Glu Asp Arg Thr Ala Ser Ala Glu Glu Ile Lys Ala Phe 245 250 255 gaa
aaa gaa ctg tct cac gca acc att gat tct aaa act ggc gat tta 816 Glu
Lys Glu Leu Ser His Ala Thr Ile Asp Ser Lys Thr Gly Asp Leu 260 265
270 ggg gac atc aat gct gag cag ctt cct ggg agg gaa cat ctt aat gaa
864 Gly Asp Ile Asn Ala Glu Gln Leu Pro Gly Arg Glu His Leu Asn Glu
275 280 285 cct ggt act aga gaa gga cag act cgt cta atc aga gat ggg
gag aaa 912 Pro Gly Thr Arg Glu Gly Gln Thr Arg Leu Ile Arg Asp Gly
Glu Lys 290 295 300 gtc gaa gcc tat cag tgg agt gtt agt gaa ggg agg
tgg ata aaa att 960 Val Glu Ala Tyr Gln Trp Ser Val Ser Glu Gly Arg
Trp Ile Lys Ile
305 310 315 320 ggt gat gtt gtt ggc tca tct ggt gct aat cag caa aca
tct gga aaa 1008 Gly Asp Val Val Gly Ser Ser Gly Ala Asn Gln Gln
Thr Ser Gly Lys 325 330 335 gtt tta tat gaa ggg aaa gaa ttt gat tat
gtt ttc tca att gat gtc 1056 Val Leu Tyr Glu Gly Lys Glu Phe Asp
Tyr Val Phe Ser Ile Asp Val 340 345 350 aat gaa ggt gga cca tca tat
aaa ttg cca tat aat acc agt gat gac 1104 Asn Glu Gly Gly Pro Ser
Tyr Lys Leu Pro Tyr Asn Thr Ser Asp Asp 355 360 365 cct tgg tta act
gca tac aac ttc tta cag aag aat gat ttg aat cct 1152 Pro Trp Leu
Thr Ala Tyr Asn Phe Leu Gln Lys Asn Asp Leu Asn Pro 370 375 380 atg
ttt ctg gat caa gta gct aaa ttt att att gat aac aca aaa ggt 1200
Met Phe Leu Asp Gln Val Ala Lys Phe Ile Ile Asp Asn Thr Lys Gly 385
390 395 400 caa atg ttg gga ctt ggg aat ccc agc ttt tca gat cca ttt
aca ggt 1248 Gln Met Leu Gly Leu Gly Asn Pro Ser Phe Ser Asp Pro
Phe Thr Gly 405 410 415 ggt ggt cgg tat gtt ccg ggc tct tcg gga tct
tct aac aca cta ccc 1296 Gly Gly Arg Tyr Val Pro Gly Ser Ser Gly
Ser Ser Asn Thr Leu Pro 420 425 430 aca gca gat cct ttt aca ggt gct
ggt cgt tat gta cca ggt tct gca 1344 Thr Ala Asp Pro Phe Thr Gly
Ala Gly Arg Tyr Val Pro Gly Ser Ala 435 440 445 agt atg gga act acc
atg gcc gga gtt gat cca ttt aca ggg aat agt 1392 Ser Met Gly Thr
Thr Met Ala Gly Val Asp Pro Phe Thr Gly Asn Ser 450 455 460 gcc tac
cga tca gct gca tct aaa aca atg aat att tat ttc cct aaa 1440 Ala
Tyr Arg Ser Ala Ala Ser Lys Thr Met Asn Ile Tyr Phe Pro Lys 465 470
475 480 aaa gag gct gtc aca ttt gac caa gca aac cct aca caa ata tta
ggt 1488 Lys Glu Ala Val Thr Phe Asp Gln Ala Asn Pro Thr Gln Ile
Leu Gly 485 490 495 aaa ctg aag gaa ctt aat gga act gca cct gaa gag
aag aag tta act 1536 Lys Leu Lys Glu Leu Asn Gly Thr Ala Pro Glu
Glu Lys Lys Leu Thr 500 505 510 gag gat gac ttg ata ctt ctt gag aag
ata ctg tct cta ata tgt aat 1584 Glu Asp Asp Leu Ile Leu Leu Glu
Lys Ile Leu Ser Leu Ile Cys Asn 515 520 525 agt tct tca gaa aaa ccc
aca gtc cag caa ctt cag att ttg tgg aaa 1632 Ser Ser Ser Glu Lys
Pro Thr Val Gln Gln Leu Gln Ile Leu Trp Lys 530 535 540 gct att aac
tgt cct gaa gat att gtc ttt cct gca ctt gac att ctt 1680 Ala Ile
Asn Cys Pro Glu Asp Ile Val Phe Pro Ala Leu Asp Ile Leu 545 550 555
560 cgg ttg tca att aaa cac ccc agt gtg aat gag aac ttc tgc aat gaa
1728 Arg Leu Ser Ile Lys His Pro Ser Val Asn Glu Asn Phe Cys Asn
Glu 565 570 575 aag gaa ggg gct cag ttc agc agt cat ctt atc aat ctt
ctg aac cct 1776 Lys Glu Gly Ala Gln Phe Ser Ser His Leu Ile Asn
Leu Leu Asn Pro 580 585 590 aaa gga aag cca gca aac cag ctg ctt gct
ctc agg act ttt tgc aat 1824 Lys Gly Lys Pro Ala Asn Gln Leu Leu
Ala Leu Arg Thr Phe Cys Asn 595 600 605 tgt ttt gtt ggc cag gca gga
caa aaa ctc atg atg tcc cag agg gaa 1872 Cys Phe Val Gly Gln Ala
Gly Gln Lys Leu Met Met Ser Gln Arg Glu 610 615 620 tca ctg atg tcc
cat gca ata gaa ctg aaa tca ggg agc aat aag aac 1920 Ser Leu Met
Ser His Ala Ile Glu Leu Lys Ser Gly Ser Asn Lys Asn 625 630 635 640
att cac att gct ctg gct aca ttg gcc ctg aac tat tct gtt tgt ttt
1968 Ile His Ile Ala Leu Ala Thr Leu Ala Leu Asn Tyr Ser Val Cys
Phe 645 650 655 cat aaa gac cat aac att gaa ggg aaa gcc caa tgt ttg
tca cta att 2016 His Lys Asp His Asn Ile Glu Gly Lys Ala Gln Cys
Leu Ser Leu Ile 660 665 670 agc aca atc ttg gaa gta gta caa gac cta
gaa gcc act ttt aga ctt 2064 Ser Thr Ile Leu Glu Val Val Gln Asp
Leu Glu Ala Thr Phe Arg Leu 675 680 685 ctt gtg gct ctt gga aca ctt
atc agt gat gat tca aat gct gta caa 2112 Leu Val Ala Leu Gly Thr
Leu Ile Ser Asp Asp Ser Asn Ala Val Gln 690 695 700 tta gcc aag tct
tta ggt gtt gat tct caa ata aaa aag tat tcc tca 2160 Leu Ala Lys
Ser Leu Gly Val Asp Ser Gln Ile Lys Lys Tyr Ser Ser 705 710 715 720
gta tca gaa cca gct aaa gta agt gaa tgc tgt aga ttt atc cta aat
2208 Val Ser Glu Pro Ala Lys Val Ser Glu Cys Cys Arg Phe Ile Leu
Asn 725 730 735 ttg ctg tag 2217 Leu Leu <210> SEQ ID NO 30
<211> LENGTH: 738 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 30 Met His Cys Met Ser Gly His
Ser Asn Phe Val Ser Cys Val Cys Ile 1 5 10 15 Ile Pro Ser Ser Asp
Ile Tyr Pro His Gly Leu Ile Ala Thr Gly Gly 20 25 30 Asn Asp His
Asn Ile Cys Ile Phe Ser Leu Asp Ser Pro Met Pro Leu 35 40 45 Tyr
Ile Leu Lys Gly His Lys Asn Thr Val Cys Ser Leu Ser Ser Gly 50 55
60 Lys Phe Gly Thr Leu Leu Ser Gly Ser Trp Asp Thr Thr Ala Lys Val
65 70 75 80 Trp Leu Asn Asp Lys Cys Met Met Thr Leu Gln Gly His Thr
Ala Ala 85 90 95 Val Trp Ala Val Lys Ile Leu Pro Glu Gln Gly Leu
Met Leu Thr Gly 100 105 110 Ser Ala Asp Lys Thr Val Lys Leu Trp Lys
Ala Gly Arg Cys Glu Arg 115 120 125 Thr Phe Ser Gly His Glu Asp Cys
Val Arg Gly Leu Ala Ile Leu Ser 130 135 140 Glu Thr Glu Phe Leu Ser
Cys Ala Asn Asp Ala Ser Ile Arg Arg Trp 145 150 155 160 Gln Ile Thr
Gly Glu Cys Leu Glu Val Tyr Tyr Gly His Thr Asn Tyr 165 170 175 Ile
Tyr Ser Ile Ser Val Phe Pro Asn Cys Arg Asp Phe Val Thr Thr 180 185
190 Ala Glu Asp Arg Ser Leu Arg Ile Trp Lys His Gly Glu Cys Ala Gln
195 200 205 Thr Ile Arg Leu Pro Ala Gln Ser Ile Trp Cys Cys Cys Val
Leu Asp 210 215 220 Asn Gly Asp Ile Val Val Gly Ala Ser Asp Gly Ile
Ile Arg Val Phe 225 230 235 240 Thr Glu Ser Glu Asp Arg Thr Ala Ser
Ala Glu Glu Ile Lys Ala Phe 245 250 255 Glu Lys Glu Leu Ser His Ala
Thr Ile Asp Ser Lys Thr Gly Asp Leu 260 265 270 Gly Asp Ile Asn Ala
Glu Gln Leu Pro Gly Arg Glu His Leu Asn Glu 275 280 285 Pro Gly Thr
Arg Glu Gly Gln Thr Arg Leu Ile Arg Asp Gly Glu Lys 290 295 300 Val
Glu Ala Tyr Gln Trp Ser Val Ser Glu Gly Arg Trp Ile Lys Ile 305 310
315 320 Gly Asp Val Val Gly Ser Ser Gly Ala Asn Gln Gln Thr Ser Gly
Lys 325 330 335 Val Leu Tyr Glu Gly Lys Glu Phe Asp Tyr Val Phe Ser
Ile Asp Val 340 345 350 Asn Glu Gly Gly Pro Ser Tyr Lys Leu Pro Tyr
Asn Thr Ser Asp Asp 355 360 365 Pro Trp Leu Thr Ala Tyr Asn Phe Leu
Gln Lys Asn Asp Leu Asn Pro 370 375 380 Met Phe Leu Asp Gln Val Ala
Lys Phe Ile Ile Asp Asn Thr Lys Gly 385 390 395 400 Gln Met Leu Gly
Leu Gly Asn Pro Ser Phe Ser Asp Pro Phe Thr Gly 405 410 415 Gly Gly
Arg Tyr Val Pro Gly Ser Ser Gly Ser Ser Asn Thr Leu Pro 420 425 430
Thr Ala Asp Pro Phe Thr Gly Ala Gly Arg Tyr Val Pro Gly Ser Ala 435
440 445 Ser Met Gly Thr Thr Met Ala Gly Val Asp Pro Phe Thr Gly Asn
Ser 450 455 460 Ala Tyr Arg Ser Ala Ala Ser Lys Thr Met Asn Ile Tyr
Phe Pro Lys 465 470 475 480 Lys Glu Ala Val Thr Phe Asp Gln Ala Asn
Pro Thr Gln Ile Leu Gly 485 490 495 Lys Leu Lys Glu Leu Asn Gly Thr
Ala Pro Glu Glu Lys Lys Leu Thr 500 505 510 Glu Asp Asp Leu Ile Leu
Leu Glu Lys Ile Leu Ser Leu Ile Cys Asn 515 520 525 Ser Ser Ser Glu
Lys Pro Thr Val Gln Gln Leu Gln Ile Leu Trp Lys 530 535 540 Ala Ile
Asn Cys Pro Glu Asp Ile Val Phe Pro Ala Leu Asp Ile Leu 545 550 555
560 Arg Leu Ser Ile Lys His Pro Ser Val Asn Glu Asn Phe Cys Asn Glu
565 570 575 Lys Glu Gly Ala Gln Phe Ser Ser His Leu Ile Asn Leu Leu
Asn Pro 580 585 590 Lys Gly Lys Pro Ala Asn Gln Leu Leu Ala Leu Arg
Thr Phe Cys Asn 595 600 605 Cys Phe Val Gly Gln Ala Gly Gln Lys Leu
Met Met Ser Gln Arg Glu 610 615 620 Ser Leu Met Ser His Ala Ile Glu
Leu Lys Ser Gly Ser Asn Lys Asn 625 630 635 640 Ile His Ile Ala Leu
Ala Thr Leu Ala Leu Asn Tyr Ser Val Cys Phe 645 650 655 His Lys Asp
His Asn Ile Glu Gly Lys Ala Gln Cys Leu Ser Leu Ile 660 665 670 Ser
Thr Ile Leu Glu Val Val Gln Asp Leu Glu Ala Thr Phe Arg Leu 675 680
685 Leu Val Ala Leu Gly Thr Leu Ile Ser Asp Asp Ser Asn Ala Val Gln
690 695 700 Leu Ala Lys Ser Leu Gly Val Asp Ser Gln Ile Lys Lys Tyr
Ser Ser 705 710 715 720
Val Ser Glu Pro Ala Lys Val Ser Glu Cys Cys Arg Phe Ile Leu Asn 725
730 735 Leu Leu <210> SEQ ID NO 31 <211> LENGTH: 911
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (27)...(743) <221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(911) <223> OTHER INFORMATION: n
= A,T,C or G <400> SEQUENCE: 31 cggcacaggg cgacggccgc tcgcca
atg tgc ctc tct gcc gtt tca ttc aag 53 Met Cys Leu Ser Ala Val Ser
Phe Lys 1 5 gga ata aga tgc tgg ctg gac aaa ctg tta ctt tgg gct ctt
aca att 101 Gly Ile Arg Cys Trp Leu Asp Lys Leu Leu Leu Trp Ala Leu
Thr Ile 10 15 20 25 tct atc aca ctt cag aat gct gca gtg gat tgt acg
agg gtg gaa aat 149 Ser Ile Thr Leu Gln Asn Ala Ala Val Asp Cys Thr
Arg Val Glu Asn 30 35 40 aac gaa tta cct tct cca aat ctg aac tca
agt atg aac gtg gtc agg 197 Asn Glu Leu Pro Ser Pro Asn Leu Asn Ser
Ser Met Asn Val Val Arg 45 50 55 atg ggc caa aat gta tct ctg tct
tgt tcc acc aag aac aca tca gta 245 Met Gly Gln Asn Val Ser Leu Ser
Cys Ser Thr Lys Asn Thr Ser Val 60 65 70 gac atc acc tat tcg ctc
ttc tgg ggt aca aaa tat cta gaa agc aag 293 Asp Ile Thr Tyr Ser Leu
Phe Trp Gly Thr Lys Tyr Leu Glu Ser Lys 75 80 85 aga aga cga ggg
gga gct gtg gat ttc cac ctg agg atc tcc aat gcc 341 Arg Arg Arg Gly
Gly Ala Val Asp Phe His Leu Arg Ile Ser Asn Ala 90 95 100 105 aac
gag tca ggc ccc tac aaa tgc aaa gtc aat gtt tcc aac ttg atg 389 Asn
Glu Ser Gly Pro Tyr Lys Cys Lys Val Asn Val Ser Asn Leu Met 110 115
120 aaa tac agt cag gat ttc aac ttc aca atg gcc aaa gat gag agc tgc
437 Lys Tyr Ser Gln Asp Phe Asn Phe Thr Met Ala Lys Asp Glu Ser Cys
125 130 135 cct tca tgc cgg ctg tca ctg ttg ctc cca ggg ctg tta ctg
ggg ata 485 Pro Ser Cys Arg Leu Ser Leu Leu Leu Pro Gly Leu Leu Leu
Gly Ile 140 145 150 ctg gta ata gtc cta gtt ctg gct tat ttg att cat
cta aaa tac aaa 533 Leu Val Ile Val Leu Val Leu Ala Tyr Leu Ile His
Leu Lys Tyr Lys 155 160 165 aaa gga aag aag act cag aga gag gac cag
tcc aag ggt tct gga gat 581 Lys Gly Lys Lys Thr Gln Arg Glu Asp Gln
Ser Lys Gly Ser Gly Asp 170 175 180 185 gcg cct gca cag gac gag ctg
tat gtc aac gcc tgc aag act cag aca 629 Ala Pro Ala Gln Asp Glu Leu
Tyr Val Asn Ala Cys Lys Thr Gln Thr 190 195 200 gag caa ccc cag gag
ata cac tat gcc act cca gtc ttc aag gag atg 677 Glu Gln Pro Gln Glu
Ile His Tyr Ala Thr Pro Val Phe Lys Glu Met 205 210 215 gca ccc atg
gaa gaa gaa ggt ggt acg gat gga aaa gct gat tac atc 725 Ala Pro Met
Glu Glu Glu Gly Gly Thr Asp Gly Lys Ala Asp Tyr Ile 220 225 230 tac
tct gaa ctc acc cac tgaagtgtga agaaactgac tgtatcccag 773 Tyr Ser
Glu Leu Thr His 235 tgtaaagact ttccagtaag ctggtgtatg agaaaatagg
aaaactcacc tggcacttaa 833 gagttccatt ctangctgan gcaggaggat
cctgagtttt gangccactt gggactacat 893 agcaagacct ggcttaaa 911
<210> SEQ ID NO 32 <211> LENGTH: 720 <212> TYPE:
DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 32
atgtgcctct ctgccgtttc attcaaggga ataagatgct ggctggacaa actgttactt
60 tgggctctta caatttctat cacacttcag aatgctgcag tggattgtac
gagggtggaa 120 aataacgaat taccttctcc aaatctgaac tcaagtatga
acgtggtcag gatgggccaa 180 aatgtatctc tgtcttgttc caccaagaac
acatcagtag acatcaccta ttcgctcttc 240 tggggtacaa aatatctaga
aagcaagaga agacgagggg gagctgtgga tttccacctg 300 aggatctcca
atgccaacga gtcaggcccc tacaaatgca aagtcaatgt ttccaacttg 360
atgaaataca gtcaggattt caacttcaca atggccaaag atgagagctg cccttcatgc
420 cggctgtcac tgttgctccc agggctgtta ctggggatac tggtaatagt
cctagttctg 480 gcttatttga ttcatctaaa atacaaaaaa ggaaagaaga
ctcagagaga ggaccagtcc 540 aagggttctg gagatgcgcc tgcacaggac
gagctgtatg tcaacgcctg caagactcag 600 acagagcaac cccaggagat
acactatgcc actccagtct tcaaggagat ggcacccatg 660 gaagaagaag
gtggtacgga tggaaaagct gattacatct actctgaact cacccactga 720
<210> SEQ ID NO 33 <211> LENGTH: 239 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: SIGNAL <222> LOCATION: (1)...(34)
<400> SEQUENCE: 33 Met Cys Leu Ser Ala Val Ser Phe Lys Gly
Ile Arg Cys Trp Leu Asp -30 -25 -20 Lys Leu Leu Leu Trp Ala Leu Thr
Ile Ser Ile Thr Leu Gln Asn Ala -15 -10 -5 Ala Val Asp Cys Thr Arg
Val Glu Asn Asn Glu Leu Pro Ser Pro Asn 1 5 10 Leu Asn Ser Ser Met
Asn Val Val Arg Met Gly Gln Asn Val Ser Leu 15 20 25 30 Ser Cys Ser
Thr Lys Asn Thr Ser Val Asp Ile Thr Tyr Ser Leu Phe 35 40 45 Trp
Gly Thr Lys Tyr Leu Glu Ser Lys Arg Arg Arg Gly Gly Ala Val 50 55
60 Asp Phe His Leu Arg Ile Ser Asn Ala Asn Glu Ser Gly Pro Tyr Lys
65 70 75 Cys Lys Val Asn Val Ser Asn Leu Met Lys Tyr Ser Gln Asp
Phe Asn 80 85 90 Phe Thr Met Ala Lys Asp Glu Ser Cys Pro Ser Cys
Arg Leu Ser Leu 95 100 105 110 Leu Leu Pro Gly Leu Leu Leu Gly Ile
Leu Val Ile Val Leu Val Leu 115 120 125 Ala Tyr Leu Ile His Leu Lys
Tyr Lys Lys Gly Lys Lys Thr Gln Arg 130 135 140 Glu Asp Gln Ser Lys
Gly Ser Gly Asp Ala Pro Ala Gln Asp Glu Leu 145 150 155 Tyr Val Asn
Ala Cys Lys Thr Gln Thr Glu Gln Pro Gln Glu Ile His 160 165 170 Tyr
Ala Thr Pro Val Phe Lys Glu Met Ala Pro Met Glu Glu Glu Gly 175 180
185 190 Gly Thr Asp Gly Lys Ala Asp Tyr Ile Tyr Ser Glu Leu Thr His
195 200 205 <210> SEQ ID NO 34 <211> LENGTH: 205
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 34 Asp Cys Thr Arg Val Glu Asn Asn Glu Leu
Pro Ser Pro Asn Leu Asn 1 5 10 15 Ser Ser Met Asn Val Val Arg Met
Gly Gln Asn Val Ser Leu Ser Cys 20 25 30 Ser Thr Lys Asn Thr Ser
Val Asp Ile Thr Tyr Ser Leu Phe Trp Gly 35 40 45 Thr Lys Tyr Leu
Glu Ser Lys Arg Arg Arg Gly Gly Ala Val Asp Phe 50 55 60 His Leu
Arg Ile Ser Asn Ala Asn Glu Ser Gly Pro Tyr Lys Cys Lys 65 70 75 80
Val Asn Val Ser Asn Leu Met Lys Tyr Ser Gln Asp Phe Asn Phe Thr 85
90 95 Met Ala Lys Asp Glu Ser Cys Pro Ser Cys Arg Leu Ser Leu Leu
Leu 100 105 110 Pro Gly Leu Leu Leu Gly Ile Leu Val Ile Val Leu Val
Leu Ala Tyr 115 120 125 Leu Ile His Leu Lys Tyr Lys Lys Gly Lys Lys
Thr Gln Arg Glu Asp 130 135 140 Gln Ser Lys Gly Ser Gly Asp Ala Pro
Ala Gln Asp Glu Leu Tyr Val 145 150 155 160 Asn Ala Cys Lys Thr Gln
Thr Glu Gln Pro Gln Glu Ile His Tyr Ala 165 170 175 Thr Pro Val Phe
Lys Glu Met Ala Pro Met Glu Glu Glu Gly Gly Thr 180 185 190 Asp Gly
Lys Ala Asp Tyr Ile Tyr Ser Glu Leu Thr His 195 200 205 <210>
SEQ ID NO 35 <211> LENGTH: 34 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <220> FEATURE: <221>
NAME/KEY: SIGNAL <222> LOCATION: (1)...(34) <400>
SEQUENCE: 35 Met Cys Leu Ser Ala Val Ser Phe Lys Gly Ile Arg Cys
Trp Leu Asp -30 -25 -20 Lys Leu Leu Leu Trp Ala Leu Thr Ile Ser Ile
Thr Leu Gln Asn Ala -15 -10 -5 Ala Val <210> SEQ ID NO 36
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 36
Asp Cys Thr Arg Val Glu Asn Asn Glu Leu Pro Ser Pro Asn Leu Asn 1 5
10 15 Ser Ser Met Asn Val Val Arg Met Gly Gln Asn Val Ser Leu Ser
Cys 20 25 30 Ser Thr Lys Asn Thr Ser Val Asp Ile Thr Tyr Ser Leu
Phe Trp Gly 35 40 45 Thr Lys Tyr Leu Glu Ser Lys Arg Arg Arg Gly
Gly Ala Val Asp Phe 50 55 60 His Leu Arg Ile Ser Asn Ala Asn Glu
Ser Gly Pro Tyr Lys Cys Lys 65 70 75 80 Val Asn Val Ser Asn Leu Met
Lys Tyr Ser Gln Asp Phe Asn Phe Thr 85 90 95 Met Ala Lys Asp Glu
Ser Cys Pro Ser Cys Arg 100 105 <210> SEQ ID NO 37
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 37 Leu Ser Leu Leu Leu Pro Gly
Leu Leu Leu Gly Ile Leu Val Ile Val 1 5 10 15 Leu Val Leu Ala Tyr
Leu Ile 20 <210> SEQ ID NO 38 <211> LENGTH: 75
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 38 His Leu Lys Tyr Lys Lys Gly Lys Lys Thr
Gln Arg Glu Asp Gln Ser 1 5 10 15 Lys Gly Ser Gly Asp Ala Pro Ala
Gln Asp Glu Leu Tyr Val Asn Ala 20 25 30 Cys Lys Thr Gln Thr Glu
Gln Pro Gln Glu Ile His Tyr Ala Thr Pro 35 40 45 Val Phe Lys Glu
Met Ala Pro Met Glu Glu Glu Gly Gly Thr Asp Gly 50 55 60 Lys Ala
Asp Tyr Ile Tyr Ser Glu Leu Thr His 65 70 75 <210> SEQ ID NO
39 <211> LENGTH: 2426 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (2)...(1117) <400> SEQUENCE: 39 c
atg gct gcg ccc gcg cga gag ccg gct ctc cgc tgc tgc atc aga ctg 49
Met Ala Ala Pro Ala Arg Glu Pro Ala Leu Arg Cys Cys Ile Arg Leu 1 5
10 15 gcg cga gtc ttc ttg ctg ctg gtg ttg gcg tgc gag gtg gcg gga
agc 97 Ala Arg Val Phe Leu Leu Leu Val Leu Ala Cys Glu Val Ala Gly
Ser 20 25 30 gat gag gcc gag gcc agg gaa ggt gcg gcg tcc ctt gcg
ggc tcg tgc 145 Asp Glu Ala Glu Ala Arg Glu Gly Ala Ala Ser Leu Ala
Gly Ser Cys 35 40 45 ggc tgc gga acg ccc cag agg gcc ggg gcc cat
ggc agc tcg gcg gcg 193 Gly Cys Gly Thr Pro Gln Arg Ala Gly Ala His
Gly Ser Ser Ala Ala 50 55 60 gcg cag cgc tac tcc cgg gag gcg aac
gcc ccg ggc ctg acc tca ggc 241 Ala Gln Arg Tyr Ser Arg Glu Ala Asn
Ala Pro Gly Leu Thr Ser Gly 65 70 75 80 ccg cga ccg ctc gcg ctc acc
aag atg gtc ccc att cct gct gga gta 289 Pro Arg Pro Leu Ala Leu Thr
Lys Met Val Pro Ile Pro Ala Gly Val 85 90 95 ttc aca atg ggc act
gat gat cct cag atc agg cag gat gga gaa gcc 337 Phe Thr Met Gly Thr
Asp Asp Pro Gln Ile Arg Gln Asp Gly Glu Ala 100 105 110 cct gcc agg
aga gtc act gtt gat ggc ttt tac atg gac gcc tat gaa 385 Pro Ala Arg
Arg Val Thr Val Asp Gly Phe Tyr Met Asp Ala Tyr Glu 115 120 125 gtc
agc aat gcg gat ttt gag aag ttt gtg aac tcg act ggc tat ttg 433 Val
Ser Asn Ala Asp Phe Glu Lys Phe Val Asn Ser Thr Gly Tyr Leu 130 135
140 aca gag gct gag aag ttt gga gac tct ttc gtc ttt gaa ggc atg ttg
481 Thr Glu Ala Glu Lys Phe Gly Asp Ser Phe Val Phe Glu Gly Met Leu
145 150 155 160 agc gag caa gtg aaa acg cat atc cac cag gca gtt gca
gct gct cca 529 Ser Glu Gln Val Lys Thr His Ile His Gln Ala Val Ala
Ala Ala Pro 165 170 175 tgg tgg ttg cct gtc aag gga gct aat tgg aga
cac cca gag ggt ccg 577 Trp Trp Leu Pro Val Lys Gly Ala Asn Trp Arg
His Pro Glu Gly Pro 180 185 190 gac tcc agt att ctg cac agg tca aat
cat ccg gtt ctc cat gtt tcc 625 Asp Ser Ser Ile Leu His Arg Ser Asn
His Pro Val Leu His Val Ser 195 200 205 tgg aac gat gct gtt gcc tac
tgc aca tgg gcg ggc aag agg ttg cct 673 Trp Asn Asp Ala Val Ala Tyr
Cys Thr Trp Ala Gly Lys Arg Leu Pro 210 215 220 act gag gca gag tgg
gaa tac agc tgt aga gga ggc ctg cag aac agg 721 Thr Glu Ala Glu Trp
Glu Tyr Ser Cys Arg Gly Gly Leu Gln Asn Arg 225 230 235 240 ctt ttc
ccc tgg ggc aac aaa ctg cag ccc aaa gga cag cat tat gcc 769 Leu Phe
Pro Trp Gly Asn Lys Leu Gln Pro Lys Gly Gln His Tyr Ala 245 250 255
aac atc tgg cag ggc aag ttt cct gtg agc aac act ggc gag gat ggc 817
Asn Ile Trp Gln Gly Lys Phe Pro Val Ser Asn Thr Gly Glu Asp Gly 260
265 270 ttc caa gga act gcc ccc gtt gat gcc ttt cct ccc aat ggc tat
ggc 865 Phe Gln Gly Thr Ala Pro Val Asp Ala Phe Pro Pro Asn Gly Tyr
Gly 275 280 285 tta tac aac ata gtg ggg aat gtg tgg gag tgg acc tca
gac tgg tgg 913 Leu Tyr Asn Ile Val Gly Asn Val Trp Glu Trp Thr Ser
Asp Trp Trp 290 295 300 act gtt cac cat tct gtt gag gaa acg ttc aac
cca aag ggt ccc act 961 Thr Val His His Ser Val Glu Glu Thr Phe Asn
Pro Lys Gly Pro Thr 305 310 315 320 tct ggg aaa gac cga gtg aag aag
ggt gga tcc tac atg tgc cat aag 1009 Ser Gly Lys Asp Arg Val Lys
Lys Gly Gly Ser Tyr Met Cys His Lys 325 330 335 tcc tat tgc tat agg
tac cgc tgt gca gct cga agc cag aac aca cca 1057 Ser Tyr Cys Tyr
Arg Tyr Arg Cys Ala Ala Arg Ser Gln Asn Thr Pro 340 345 350 gat agc
tct gca tcc aac ctg gga ttc cga tgt gca gcc gac cac ctg 1105 Asp
Ser Ser Ala Ser Asn Leu Gly Phe Arg Cys Ala Ala Asp His Leu 355 360
365 ccc acc gca gac tgacagccaa gaggagcttt tccagattca agaaggcgtt
1157 Pro Thr Ala Asp 370 tcttactcgc agctggcctc ccctggaaat
ctgaactgat ctcatgtaaa gtattcccat 1217 ctacggagga ttccatgtcc
acccagtggc caaaggaact gtctcgcgtg accaaatggc 1277 tggcgagtgt
cagcacgtgt gctttattgt gtggtgtatc tttggggatc atcgccatgt 1337
tttactttga aagccttttg aagaggagag agccgagaac caggaagtcc tggccagact
1397 ctgccacagg gtcagaccct ggagtccagc accttgtctg ccttgacctc
tgtctcctca 1457 tgaaatgagg gatggtcaac gtgatctttg aggctctctc
caactctatt tgaactagca 1517 gattctattc gaactagcag agtgtattgt
gattgcatag tgagaattta tgacagatta 1577 ttttttagct atttttttgc
catgtgtgaa tcttgagtaa tactaatcat ataaggcgag 1637 agttatctta
catattattt tcagaaaagg gtggggtttg agtcttttat attcatactg 1697
cactttgttc tttcaaggaa atcagtgtct tttacattgt tgtgacaaat cccattggga
1757 cagcgagggg acacttaagt ttggagttct gaacacacag gaatgcctgt
ggagtgactc 1817 tactgtcctt tttcttttga cattaagtgc ctttggctca
gagggacagt ttgaagcctt 1877 gtttcccctt tgcccccaag ccttcaaaga
atgtgaaata tgtactaatt agggaaacca 1937 tttaattcta ggtctttggg
tgttgaggtt ttgtcagatg gtatgaattg tattgtaatg 1997 ctaaatctgg
tacctgaagg tctaggcctg tgagtgaatt ctcacattta caagattttg 2057
ttgtgcaaac cttgttcctt aatttaaaac tattggttaa ataaaattgg ctacagccaa
2117 ttactggagg gattagaggt aggtgggttt tgagtgtggt tgggtatgga
gagagagagg 2177 agagatcaag gagagaagca ggaagaagag gataggagga
gctgccatga agaagatgga 2237 ccataagcct ttgtccagag gaaactccca
agtatctggg aacaccgctg tgaggcaacc 2297 aggccagcag ttagaaaagt
agattagggg ctacccccag taattgtcaa agccaaataa 2357 aatatcaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aacaaaaaaa 2417
aaaaaaaaa 2426 <210> SEQ ID NO 40 <211> LENGTH: 1119
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<400> SEQUENCE: 40 atggctgcgc ccgcgcgaga gccggctctc
cgctgctgca tcagactggc gcgagtcttc 60 ttgctgctgg tgttggcgtg
cgaggtggcg ggaagcgatg aggccgaggc cagggaaggt 120 gcggcgtccc
ttgcgggctc gtgcggctgc ggaacgcccc agagggccgg ggcccatggc 180
agctcggcgg cggcgcagcg ctactcccgg gaggcgaacg ccccgggcct gacctcaggc
240 ccgcgaccgc tcgcgctcac caagatggtc cccattcctg ctggagtatt
cacaatgggc 300 actgatgatc ctcagatcag gcaggatgga gaagcccctg
ccaggagagt cactgttgat 360 ggcttttaca tggacgccta tgaagtcagc
aatgcggatt ttgagaagtt tgtgaactcg 420 actggctatt tgacagaggc
tgagaagttt ggagactctt tcgtctttga aggcatgttg 480 agcgagcaag
tgaaaacgca tatccaccag gcagttgcag ctgctccatg gtggttgcct 540
gtcaagggag ctaattggag acacccagag ggtccggact ccagtattct gcacaggtca
600 aatcatccgg ttctccatgt ttcctggaac gatgctgttg cctactgcac
atgggcgggc 660 aagaggttgc ctactgaggc agagtgggaa tacagctgta
gaggaggcct gcagaacagg 720 cttttcccct ggggcaacaa actgcagccc
aaaggacagc attatgccaa catctggcag 780 ggcaagtttc ctgtgagcaa
cactggcgag gatggcttcc aaggaactgc ccccgttgat 840 gcctttcctc
ccaatggcta tggcttatac aacatagtgg ggaatgtgtg ggagtggacc 900
tcagactggt ggactgttca ccattctgtt gaggaaacgt tcaacccaaa gggtcccact
960 tctgggaaag accgagtgaa gaagggtgga tcctacatgt gccataagtc
ctattgctat 1020
aggtaccgct gtgcagctcg aagccagaac acaccagata gctctgcatc caacctggga
1080 ttccgatgtg cagccgacca cctgcccacc gcagactga 1119 <210>
SEQ ID NO 41 <211> LENGTH: 372 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <220> FEATURE: <221>
NAME/KEY: SIGNAL <222> LOCATION: (1)...(31) <400>
SEQUENCE: 41 Met Ala Ala Pro Ala Arg Glu Pro Ala Leu Arg Cys Cys
Ile Arg Leu -30 -25 -20 Ala Arg Val Phe Leu Leu Leu Val Leu Ala Cys
Glu Val Ala Gly Ser -15 -10 -5 1 Asp Glu Ala Glu Ala Arg Glu Gly
Ala Ala Ser Leu Ala Gly Ser Cys 5 10 15 Gly Cys Gly Thr Pro Gln Arg
Ala Gly Ala His Gly Ser Ser Ala Ala 20 25 30 Ala Gln Arg Tyr Ser
Arg Glu Ala Asn Ala Pro Gly Leu Thr Ser Gly 35 40 45 Pro Arg Pro
Leu Ala Leu Thr Lys Met Val Pro Ile Pro Ala Gly Val 50 55 60 65 Phe
Thr Met Gly Thr Asp Asp Pro Gln Ile Arg Gln Asp Gly Glu Ala 70 75
80 Pro Ala Arg Arg Val Thr Val Asp Gly Phe Tyr Met Asp Ala Tyr Glu
85 90 95 Val Ser Asn Ala Asp Phe Glu Lys Phe Val Asn Ser Thr Gly
Tyr Leu 100 105 110 Thr Glu Ala Glu Lys Phe Gly Asp Ser Phe Val Phe
Glu Gly Met Leu 115 120 125 Ser Glu Gln Val Lys Thr His Ile His Gln
Ala Val Ala Ala Ala Pro 130 135 140 145 Trp Trp Leu Pro Val Lys Gly
Ala Asn Trp Arg His Pro Glu Gly Pro 150 155 160 Asp Ser Ser Ile Leu
His Arg Ser Asn His Pro Val Leu His Val Ser 165 170 175 Trp Asn Asp
Ala Val Ala Tyr Cys Thr Trp Ala Gly Lys Arg Leu Pro 180 185 190 Thr
Glu Ala Glu Trp Glu Tyr Ser Cys Arg Gly Gly Leu Gln Asn Arg 195 200
205 Leu Phe Pro Trp Gly Asn Lys Leu Gln Pro Lys Gly Gln His Tyr Ala
210 215 220 225 Asn Ile Trp Gln Gly Lys Phe Pro Val Ser Asn Thr Gly
Glu Asp Gly 230 235 240 Phe Gln Gly Thr Ala Pro Val Asp Ala Phe Pro
Pro Asn Gly Tyr Gly 245 250 255 Leu Tyr Asn Ile Val Gly Asn Val Trp
Glu Trp Thr Ser Asp Trp Trp 260 265 270 Thr Val His His Ser Val Glu
Glu Thr Phe Asn Pro Lys Gly Pro Thr 275 280 285 Ser Gly Lys Asp Arg
Val Lys Lys Gly Gly Ser Tyr Met Cys His Lys 290 295 300 305 Ser Tyr
Cys Tyr Arg Tyr Arg Cys Ala Ala Arg Ser Gln Asn Thr Pro 310 315 320
Asp Ser Ser Ala Ser Asn Leu Gly Phe Arg Cys Ala Ala Asp His Leu 325
330 335 Pro Thr Ala Asp 340 <210> SEQ ID NO 42 <211>
LENGTH: 341 <212> TYPE: PRT <213> ORGANISM: Mus
musculus <400> SEQUENCE: 42 Ser Asp Glu Ala Glu Ala Arg Glu
Gly Ala Ala Ser Leu Ala Gly Ser 1 5 10 15 Cys Gly Cys Gly Thr Pro
Gln Arg Ala Gly Ala His Gly Ser Ser Ala 20 25 30 Ala Ala Gln Arg
Tyr Ser Arg Glu Ala Asn Ala Pro Gly Leu Thr Ser 35 40 45 Gly Pro
Arg Pro Leu Ala Leu Thr Lys Met Val Pro Ile Pro Ala Gly 50 55 60
Val Phe Thr Met Gly Thr Asp Asp Pro Gln Ile Arg Gln Asp Gly Glu 65
70 75 80 Ala Pro Ala Arg Arg Val Thr Val Asp Gly Phe Tyr Met Asp
Ala Tyr 85 90 95 Glu Val Ser Asn Ala Asp Phe Glu Lys Phe Val Asn
Ser Thr Gly Tyr 100 105 110 Leu Thr Glu Ala Glu Lys Phe Gly Asp Ser
Phe Val Phe Glu Gly Met 115 120 125 Leu Ser Glu Gln Val Lys Thr His
Ile His Gln Ala Val Ala Ala Ala 130 135 140 Pro Trp Trp Leu Pro Val
Lys Gly Ala Asn Trp Arg His Pro Glu Gly 145 150 155 160 Pro Asp Ser
Ser Ile Leu His Arg Ser Asn His Pro Val Leu His Val 165 170 175 Ser
Trp Asn Asp Ala Val Ala Tyr Cys Thr Trp Ala Gly Lys Arg Leu 180 185
190 Pro Thr Glu Ala Glu Trp Glu Tyr Ser Cys Arg Gly Gly Leu Gln Asn
195 200 205 Arg Leu Phe Pro Trp Gly Asn Lys Leu Gln Pro Lys Gly Gln
His Tyr 210 215 220 Ala Asn Ile Trp Gln Gly Lys Phe Pro Val Ser Asn
Thr Gly Glu Asp 225 230 235 240 Gly Phe Gln Gly Thr Ala Pro Val Asp
Ala Phe Pro Pro Asn Gly Tyr 245 250 255 Gly Leu Tyr Asn Ile Val Gly
Asn Val Trp Glu Trp Thr Ser Asp Trp 260 265 270 Trp Thr Val His His
Ser Val Glu Glu Thr Phe Asn Pro Lys Gly Pro 275 280 285 Thr Ser Gly
Lys Asp Arg Val Lys Lys Gly Gly Ser Tyr Met Cys His 290 295 300 Lys
Ser Tyr Cys Tyr Arg Tyr Arg Cys Ala Ala Arg Ser Gln Asn Thr 305 310
315 320 Pro Asp Ser Ser Ala Ser Asn Leu Gly Phe Arg Cys Ala Ala Asp
His 325 330 335 Leu Pro Thr Ala Asp 340 <210> SEQ ID NO 43
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <220> FEATURE: <221> NAME/KEY: SIGNAL
<222> LOCATION: (1)...(31) <400> SEQUENCE: 43 Met Ala
Ala Pro Ala Arg Glu Pro Ala Leu Arg Cys Cys Ile Arg Leu -30 -25 -20
Ala Arg Val Phe Leu Leu Leu Val Leu Ala Cys Glu Val Ala Gly -15 -10
-5 <210> SEQ ID NO 44 <211> LENGTH: 2811 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (3)...(2567)
<400> SEQUENCE: 44 ct cgg ccc gct cgg cgc gcc cct tcc cag ccg
ccc ttc cgt acc ggc 47 Arg Pro Ala Arg Arg Ala Pro Ser Gln Pro Pro
Phe Arg Thr Gly 1 5 10 15 tct cgg gct ctt ccg gtc tcc ggc cgc ccc
tta cct gca ggc tct tct 95 Ser Arg Ala Leu Pro Val Ser Gly Arg Pro
Leu Pro Ala Gly Ser Ser 20 25 30 ccc gcc gcg gcc cgg cgc tct ccg
agt cgc ccc tgc gga ctg gtc tcg 143 Pro Ala Ala Ala Arg Arg Ser Pro
Ser Arg Pro Cys Gly Leu Val Ser 35 40 45 cac agt gcc tgg gca ccg
ggc gcc aga cag aca ctg gcc atg acg agc 191 His Ser Ala Trp Ala Pro
Gly Ala Arg Gln Thr Leu Ala Met Thr Ser 50 55 60 ggc gca acc agg
tac cgg ctg agc tgc tcg ctc cgg ggc cac gag ctg 239 Gly Ala Thr Arg
Tyr Arg Leu Ser Cys Ser Leu Arg Gly His Glu Leu 65 70 75 gac gta
cgg ggc ctg gtg tgc tgc gcc tat ccg ccg gga gcc ttt gtg 287 Asp Val
Arg Gly Leu Val Cys Cys Ala Tyr Pro Pro Gly Ala Phe Val 80 85 90 95
tcc gtg tcc cga gac cgc acc acc cgc ctc tgg gcc cca gac agt cca 335
Ser Val Ser Arg Asp Arg Thr Thr Arg Leu Trp Ala Pro Asp Ser Pro 100
105 110 aac agg agc ttt aca gaa atg cac tgt atg agt ggc cat tcc aat
ttt 383 Asn Arg Ser Phe Thr Glu Met His Cys Met Ser Gly His Ser Asn
Phe 115 120 125 gta tct tgt gta tgc atc ata ccc tca agt gac atc tac
cct cat ggc 431 Val Ser Cys Val Cys Ile Ile Pro Ser Ser Asp Ile Tyr
Pro His Gly 130 135 140 cta att gcc acc ggt gga aat gac cac aat ata
tgc att ttc tca ctg 479 Leu Ile Ala Thr Gly Gly Asn Asp His Asn Ile
Cys Ile Phe Ser Leu 145 150 155 gac agt cca atg cca ctt tat att cta
aaa ggc cac aaa aat act gtt 527 Asp Ser Pro Met Pro Leu Tyr Ile Leu
Lys Gly His Lys Asn Thr Val 160 165 170 175 tgt agt cta tca tct gga
aaa ttt ggg aca tta ctt agt ggt tca tgg 575 Cys Ser Leu Ser Ser Gly
Lys Phe Gly Thr Leu Leu Ser Gly Ser Trp 180 185 190 gac acc act gct
aaa gtc tgg ctg aat gac aag tgc atg atg acc ttg 623 Asp Thr Thr Ala
Lys Val Trp Leu Asn Asp Lys Cys Met Met Thr Leu 195 200 205 cag ggt
cat aca gct gca gtg tgg gcg gta aag atc tta cct gaa cag 671 Gln Gly
His Thr Ala Ala Val Trp Ala Val Lys Ile Leu Pro Glu Gln 210 215 220
ggc tta atg ttg act gga tca gca gac aag act gtt aaa ctg tgg aag 719
Gly Leu Met Leu Thr Gly Ser Ala Asp Lys Thr Val Lys Leu Trp Lys 225
230 235 gct gga aga tgt gag agg act ttt tca ggg cat gaa gac tgt gta
aga 767
Ala Gly Arg Cys Glu Arg Thr Phe Ser Gly His Glu Asp Cys Val Arg 240
245 250 255 ggt ttg gca att ttg agt gaa aca gaa ttt ctt tcc tgt gca
aat gat 815 Gly Leu Ala Ile Leu Ser Glu Thr Glu Phe Leu Ser Cys Ala
Asn Asp 260 265 270 gct agt att aga agg tgg caa atc act ggc gag tgt
ctt gaa gta tat 863 Ala Ser Ile Arg Arg Trp Gln Ile Thr Gly Glu Cys
Leu Glu Val Tyr 275 280 285 tat gga cat aca aat tat att tat agc ata
tcc gtt ttt cca aat tgt 911 Tyr Gly His Thr Asn Tyr Ile Tyr Ser Ile
Ser Val Phe Pro Asn Cys 290 295 300 aga gac ttt gtg aca aca gca gag
gac aga tct ctg aga atc tgg aaa 959 Arg Asp Phe Val Thr Thr Ala Glu
Asp Arg Ser Leu Arg Ile Trp Lys 305 310 315 cat ggg gaa tgt gct caa
act atc cga ctt cca gct cag tct ata tgg 1007 His Gly Glu Cys Ala
Gln Thr Ile Arg Leu Pro Ala Gln Ser Ile Trp 320 325 330 335 tgc tgc
tgt gtg ctc gac aat ggt gac att gtg gtt ggt gcg agt gat 1055 Cys
Cys Cys Val Leu Asp Asn Gly Asp Ile Val Val Gly Ala Ser Asp 340 345
350 ggc att att aga gtg ttt aca gaa tca gaa gat cga aca gca agt gct
1103 Gly Ile Ile Arg Val Phe Thr Glu Ser Glu Asp Arg Thr Ala Ser
Ala 355 360 365 gaa gaa atc aag gct ttt gaa aaa gaa ctg tct cac gca
acc att gat 1151 Glu Glu Ile Lys Ala Phe Glu Lys Glu Leu Ser His
Ala Thr Ile Asp 370 375 380 tct aaa act ggc gat tta ggg gac atc aat
gct gag cag ctt cct ggg 1199 Ser Lys Thr Gly Asp Leu Gly Asp Ile
Asn Ala Glu Gln Leu Pro Gly 385 390 395 agg gaa cat ctt aat gaa cct
ggt act aga gaa gga cag act cgt cta 1247 Arg Glu His Leu Asn Glu
Pro Gly Thr Arg Glu Gly Gln Thr Arg Leu 400 405 410 415 atc aga gat
ggg gag aaa gtc gaa gcc tat cag tgg agt gtt agt gaa 1295 Ile Arg
Asp Gly Glu Lys Val Glu Ala Tyr Gln Trp Ser Val Ser Glu 420 425 430
ggg agg tgg ata aaa att ggt gat gtt gtt ggc tca tct ggt gct aat
1343 Gly Arg Trp Ile Lys Ile Gly Asp Val Val Gly Ser Ser Gly Ala
Asn 435 440 445 cag caa aca tct gga aaa gtt tta tat gaa ggg aaa gaa
ttt gat tat 1391 Gln Gln Thr Ser Gly Lys Val Leu Tyr Glu Gly Lys
Glu Phe Asp Tyr 450 455 460 gtt ttc tca att gat gtc aat gaa ggt gga
cca tca tat aaa ttg cca 1439 Val Phe Ser Ile Asp Val Asn Glu Gly
Gly Pro Ser Tyr Lys Leu Pro 465 470 475 tat aat acc agt gat gac cct
tgg tta act gca tac aac ttc tta cag 1487 Tyr Asn Thr Ser Asp Asp
Pro Trp Leu Thr Ala Tyr Asn Phe Leu Gln 480 485 490 495 aag aat gat
ttg aat cct atg ttt ctg gat caa gta gct aaa ttt att 1535 Lys Asn
Asp Leu Asn Pro Met Phe Leu Asp Gln Val Ala Lys Phe Ile 500 505 510
att gat aac aca aaa ggt caa atg ttg gga ctt ggg aat ccc agc ttt
1583 Ile Asp Asn Thr Lys Gly Gln Met Leu Gly Leu Gly Asn Pro Ser
Phe 515 520 525 tca gat cca ttt aca ggt ggt ggt cgg tat gtt ccg ggc
tct tcg gga 1631 Ser Asp Pro Phe Thr Gly Gly Gly Arg Tyr Val Pro
Gly Ser Ser Gly 530 535 540 tct tct aac aca cta ccc aca gca gat cct
ttt aca ggt gct ggt cgt 1679 Ser Ser Asn Thr Leu Pro Thr Ala Asp
Pro Phe Thr Gly Ala Gly Arg 545 550 555 tat gta cca ggt tct gca agt
atg gga act acc atg gcc gga gtt gat 1727 Tyr Val Pro Gly Ser Ala
Ser Met Gly Thr Thr Met Ala Gly Val Asp 560 565 570 575 cca ttt aca
ggg aat agt gcc tac cga tca gct gca tct aaa aca atg 1775 Pro Phe
Thr Gly Asn Ser Ala Tyr Arg Ser Ala Ala Ser Lys Thr Met 580 585 590
aat att tat ttc cct aaa aaa gag gct gtc aca ttt gac caa gca aac
1823 Asn Ile Tyr Phe Pro Lys Lys Glu Ala Val Thr Phe Asp Gln Ala
Asn 595 600 605 cct aca caa ata tta ggt aaa ctg aag gaa ctt aat gga
act gca cct 1871 Pro Thr Gln Ile Leu Gly Lys Leu Lys Glu Leu Asn
Gly Thr Ala Pro 610 615 620 gaa gag aag aag tta act gag gat gac ttg
ata ctt ctt gag aag ata 1919 Glu Glu Lys Lys Leu Thr Glu Asp Asp
Leu Ile Leu Leu Glu Lys Ile 625 630 635 ctg tct cta ata tgt aat agt
tct tca gaa aaa ccc aca gtc cag caa 1967 Leu Ser Leu Ile Cys Asn
Ser Ser Ser Glu Lys Pro Thr Val Gln Gln 640 645 650 655 ctt cag att
ttg tgg aaa gct att aac tgt cct gaa gat att gtc ttt 2015 Leu Gln
Ile Leu Trp Lys Ala Ile Asn Cys Pro Glu Asp Ile Val Phe 660 665 670
cct gca ctt gac att ctt cgg ttg tca att aaa cac ccc agt gtg aat
2063 Pro Ala Leu Asp Ile Leu Arg Leu Ser Ile Lys His Pro Ser Val
Asn 675 680 685 gag aac ttc tgc aat gaa aag gaa ggg gct cag ttc agc
agt cat ctt 2111 Glu Asn Phe Cys Asn Glu Lys Glu Gly Ala Gln Phe
Ser Ser His Leu 690 695 700 atc aat ctt ctg aac cct aaa gga aag cca
gca aac cag ctg ctt gct 2159 Ile Asn Leu Leu Asn Pro Lys Gly Lys
Pro Ala Asn Gln Leu Leu Ala 705 710 715 ctc agg act ttt tgc aat tgt
ttt gtt ggc cag gca gga caa aaa ctc 2207 Leu Arg Thr Phe Cys Asn
Cys Phe Val Gly Gln Ala Gly Gln Lys Leu 720 725 730 735 atg atg tcc
cag agg gaa tca ctg atg tcc cat gca ata gaa ctg aaa 2255 Met Met
Ser Gln Arg Glu Ser Leu Met Ser His Ala Ile Glu Leu Lys 740 745 750
tca ggg agc aat aag aac att cac att gct ctg gct aca ttg gcc ctg
2303 Ser Gly Ser Asn Lys Asn Ile His Ile Ala Leu Ala Thr Leu Ala
Leu 755 760 765 aac tat tct gtt tgt ttt cat aaa gac cat aac att gaa
ggg aaa gcc 2351 Asn Tyr Ser Val Cys Phe His Lys Asp His Asn Ile
Glu Gly Lys Ala 770 775 780 caa tgt ttg tca cta att agc aca atc ttg
gaa gta gta caa gac cta 2399 Gln Cys Leu Ser Leu Ile Ser Thr Ile
Leu Glu Val Val Gln Asp Leu 785 790 795 gaa gcc act ttt aga ctt ctt
gtg gct ctt gga aca ctt atc agt gat 2447 Glu Ala Thr Phe Arg Leu
Leu Val Ala Leu Gly Thr Leu Ile Ser Asp 800 805 810 815 gat tca aat
gct gta caa tta gcc aag tct tta ggt gtt gat tct caa 2495 Asp Ser
Asn Ala Val Gln Leu Ala Lys Ser Leu Gly Val Asp Ser Gln 820 825 830
ata aaa aag tat tcc tca gta tca gaa cca gct aaa gta agt gaa tgc
2543 Ile Lys Lys Tyr Ser Ser Val Ser Glu Pro Ala Lys Val Ser Glu
Cys 835 840 845 tgt aga ttt atc cta aat ttg ctg tagcagtggg
gaagagggac ggatattttt 2597 Cys Arg Phe Ile Leu Asn Leu Leu 850 855
aattgattag tgtttttttc ctcacatttg acatgactga taacagataa ttaaaaaaag
2657 agaatacggt ggattaagta aaattttaca tcttgtaaag tggtggggag
gggaaacaga 2717 aataaaattt ttgcactgct gaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 2777 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa
2811 <210> SEQ ID NO 45 <211> LENGTH: 855 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
45 Arg Pro Ala Arg Arg Ala Pro Ser Gln Pro Pro Phe Arg Thr Gly Ser
1 5 10 15 Arg Ala Leu Pro Val Ser Gly Arg Pro Leu Pro Ala Gly Ser
Ser Pro 20 25 30 Ala Ala Ala Arg Arg Ser Pro Ser Arg Pro Cys Gly
Leu Val Ser His 35 40 45 Ser Ala Trp Ala Pro Gly Ala Arg Gln Thr
Leu Ala Met Thr Ser Gly 50 55 60 Ala Thr Arg Tyr Arg Leu Ser Cys
Ser Leu Arg Gly His Glu Leu Asp 65 70 75 80 Val Arg Gly Leu Val Cys
Cys Ala Tyr Pro Pro Gly Ala Phe Val Ser 85 90 95 Val Ser Arg Asp
Arg Thr Thr Arg Leu Trp Ala Pro Asp Ser Pro Asn 100 105 110 Arg Ser
Phe Thr Glu Met His Cys Met Ser Gly His Ser Asn Phe Val 115 120 125
Ser Cys Val Cys Ile Ile Pro Ser Ser Asp Ile Tyr Pro His Gly Leu 130
135 140 Ile Ala Thr Gly Gly Asn Asp His Asn Ile Cys Ile Phe Ser Leu
Asp 145 150 155 160 Ser Pro Met Pro Leu Tyr Ile Leu Lys Gly His Lys
Asn Thr Val Cys 165 170 175 Ser Leu Ser Ser Gly Lys Phe Gly Thr Leu
Leu Ser Gly Ser Trp Asp 180 185 190 Thr Thr Ala Lys Val Trp Leu Asn
Asp Lys Cys Met Met Thr Leu Gln 195 200 205 Gly His Thr Ala Ala Val
Trp Ala Val Lys Ile Leu Pro Glu Gln Gly 210 215 220 Leu Met Leu Thr
Gly Ser Ala Asp Lys Thr Val Lys Leu Trp Lys Ala 225 230 235 240 Gly
Arg Cys Glu Arg Thr Phe Ser Gly His Glu Asp Cys Val Arg Gly 245 250
255 Leu Ala Ile Leu Ser Glu Thr Glu Phe Leu Ser Cys Ala Asn Asp Ala
260 265 270 Ser Ile Arg Arg Trp Gln Ile Thr Gly Glu Cys Leu Glu Val
Tyr Tyr 275 280 285 Gly His Thr Asn Tyr Ile Tyr Ser Ile Ser Val Phe
Pro Asn Cys Arg 290 295 300 Asp Phe Val Thr Thr Ala Glu Asp Arg Ser
Leu Arg Ile Trp Lys His 305 310 315 320 Gly Glu Cys Ala Gln Thr Ile
Arg Leu Pro Ala Gln Ser Ile Trp Cys 325 330 335 Cys Cys Val Leu Asp
Asn Gly Asp Ile Val Val Gly Ala Ser Asp Gly 340 345 350 Ile Ile Arg
Val Phe Thr Glu Ser Glu Asp Arg Thr Ala Ser Ala Glu 355 360 365 Glu
Ile Lys Ala Phe Glu Lys Glu Leu Ser His Ala Thr Ile Asp Ser 370 375
380 Lys Thr Gly Asp Leu Gly Asp Ile Asn Ala Glu Gln Leu Pro Gly Arg
385 390 395 400 Glu His Leu Asn Glu Pro Gly Thr Arg Glu Gly Gln Thr
Arg Leu Ile 405 410 415 Arg Asp Gly Glu Lys Val Glu Ala Tyr Gln Trp
Ser Val Ser Glu Gly 420 425 430
Arg Trp Ile Lys Ile Gly Asp Val Val Gly Ser Ser Gly Ala Asn Gln 435
440 445 Gln Thr Ser Gly Lys Val Leu Tyr Glu Gly Lys Glu Phe Asp Tyr
Val 450 455 460 Phe Ser Ile Asp Val Asn Glu Gly Gly Pro Ser Tyr Lys
Leu Pro Tyr 465 470 475 480 Asn Thr Ser Asp Asp Pro Trp Leu Thr Ala
Tyr Asn Phe Leu Gln Lys 485 490 495 Asn Asp Leu Asn Pro Met Phe Leu
Asp Gln Val Ala Lys Phe Ile Ile 500 505 510 Asp Asn Thr Lys Gly Gln
Met Leu Gly Leu Gly Asn Pro Ser Phe Ser 515 520 525 Asp Pro Phe Thr
Gly Gly Gly Arg Tyr Val Pro Gly Ser Ser Gly Ser 530 535 540 Ser Asn
Thr Leu Pro Thr Ala Asp Pro Phe Thr Gly Ala Gly Arg Tyr 545 550 555
560 Val Pro Gly Ser Ala Ser Met Gly Thr Thr Met Ala Gly Val Asp Pro
565 570 575 Phe Thr Gly Asn Ser Ala Tyr Arg Ser Ala Ala Ser Lys Thr
Met Asn 580 585 590 Ile Tyr Phe Pro Lys Lys Glu Ala Val Thr Phe Asp
Gln Ala Asn Pro 595 600 605 Thr Gln Ile Leu Gly Lys Leu Lys Glu Leu
Asn Gly Thr Ala Pro Glu 610 615 620 Glu Lys Lys Leu Thr Glu Asp Asp
Leu Ile Leu Leu Glu Lys Ile Leu 625 630 635 640 Ser Leu Ile Cys Asn
Ser Ser Ser Glu Lys Pro Thr Val Gln Gln Leu 645 650 655 Gln Ile Leu
Trp Lys Ala Ile Asn Cys Pro Glu Asp Ile Val Phe Pro 660 665 670 Ala
Leu Asp Ile Leu Arg Leu Ser Ile Lys His Pro Ser Val Asn Glu 675 680
685 Asn Phe Cys Asn Glu Lys Glu Gly Ala Gln Phe Ser Ser His Leu Ile
690 695 700 Asn Leu Leu Asn Pro Lys Gly Lys Pro Ala Asn Gln Leu Leu
Ala Leu 705 710 715 720 Arg Thr Phe Cys Asn Cys Phe Val Gly Gln Ala
Gly Gln Lys Leu Met 725 730 735 Met Ser Gln Arg Glu Ser Leu Met Ser
His Ala Ile Glu Leu Lys Ser 740 745 750 Gly Ser Asn Lys Asn Ile His
Ile Ala Leu Ala Thr Leu Ala Leu Asn 755 760 765 Tyr Ser Val Cys Phe
His Lys Asp His Asn Ile Glu Gly Lys Ala Gln 770 775 780 Cys Leu Ser
Leu Ile Ser Thr Ile Leu Glu Val Val Gln Asp Leu Glu 785 790 795 800
Ala Thr Phe Arg Leu Leu Val Ala Leu Gly Thr Leu Ile Ser Asp Asp 805
810 815 Ser Asn Ala Val Gln Leu Ala Lys Ser Leu Gly Val Asp Ser Gln
Ile 820 825 830 Lys Lys Tyr Ser Ser Val Ser Glu Pro Ala Lys Val Ser
Glu Cys Cys 835 840 845 Arg Phe Ile Leu Asn Leu Leu 850 855
<210> SEQ ID NO 46 <211> LENGTH: 57 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 46 Gly
Gln Asn Val Ser Leu Ser Cys Ser Thr Lys Asn Thr Ser Val Asp 1 5 10
15 Ile Thr Tyr Ser Leu Phe Trp Gly Thr Lys Tyr Leu Glu Ser Lys Arg
20 25 30 Arg Arg Gly Gly Ala Val Asp Phe His Leu Arg Ile Ser Asn
Ala Asn 35 40 45 Glu Ser Gly Pro Tyr Lys Cys Lys Val 50 55
<210> SEQ ID NO 47 <211> LENGTH: 252 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 47 Thr
Glu Ser Glu Asp Arg Thr Ala Ser Ala Glu Glu Ile Lys Ala Phe 1 5 10
15 Glu Lys Glu Leu Ser His Ala Thr Ile Asp Ser Lys Thr Gly Asp Leu
20 25 30 Gly Asp Ile Asn Ala Glu Gln Leu Pro Gly Arg Glu His Leu
Asn Glu 35 40 45 Pro Gly Thr Arg Glu Gly Gln Thr Arg Leu Ile Arg
Asp Gly Glu Lys 50 55 60 Val Glu Ala Tyr Gln Trp Ser Val Ser Glu
Gly Arg Trp Ile Lys Ile 65 70 75 80 Gly Asp Val Val Gly Ser Ser Gly
Ala Asn Gln Gln Thr Ser Gly Lys 85 90 95 Val Leu Tyr Glu Gly Lys
Glu Phe Asp Tyr Val Phe Ser Ile Asp Val 100 105 110 Asn Glu Gly Gly
Pro Ser Tyr Lys Leu Pro Tyr Asn Thr Ser Asp Asp 115 120 125 Pro Trp
Leu Thr Ala Tyr Asn Phe Leu Gln Lys Asn Asp Leu Asn Pro 130 135 140
Met Phe Leu Asp Gln Val Ala Lys Phe Ile Ile Asp Asn Thr Lys Gly 145
150 155 160 Gln Met Leu Gly Leu Gly Asn Pro Ser Phe Ser Asp Pro Phe
Thr Gly 165 170 175 Gly Gly Arg Tyr Val Pro Gly Ser Ser Gly Ser Ser
Asn Thr Leu Pro 180 185 190 Thr Ala Asp Pro Phe Thr Gly Ala Gly Arg
Tyr Val Pro Gly Ser Ala 195 200 205 Ser Met Gly Thr Thr Met Ala Gly
Val Asp Pro Phe Thr Gly Asn Ser 210 215 220 Ala Tyr Arg Ser Ala Ala
Ser Lys Thr Met Asn Ile Tyr Phe Pro Lys 225 230 235 240 Lys Glu Ala
Val Thr Phe Asp Gln Ala Asn Pro Thr 245 250 <210> SEQ ID NO
48 <211> LENGTH: 231 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 48 Ser Asp Pro Phe Thr
Gly Gly Gly Arg Tyr Val Pro Gly Ser Ser Gly 1 5 10 15 Ser Ser Asn
Thr Leu Pro Thr Ala Asp Pro Phe Thr Gly Ala Gly Arg 20 25 30 Tyr
Val Pro Gly Ser Ala Ser Met Gly Thr Thr Met Ala Gly Val Asp 35 40
45 Pro Phe Thr Gly Asn Ser Ala Tyr Arg Ser Ala Ala Ser Lys Thr Met
50 55 60 Asn Ile Tyr Phe Pro Lys Lys Glu Ala Val Thr Phe Asp Gln
Ala Asn 65 70 75 80 Pro Thr Gln Ile Leu Gly Lys Leu Lys Glu Leu Asn
Gly Thr Ala Pro 85 90 95 Glu Glu Lys Lys Leu Thr Glu Asp Asp Leu
Ile Leu Leu Glu Lys Ile 100 105 110 Leu Ser Leu Ile Cys Asn Ser Ser
Ser Glu Lys Pro Thr Val Gln Gln 115 120 125 Leu Gln Ile Leu Trp Lys
Ala Ile Asn Cys Pro Glu Asp Ile Val Phe 130 135 140 Pro Ala Leu Asp
Ile Leu Arg Leu Ser Ile Lys His Pro Ser Val Asn 145 150 155 160 Glu
Asn Phe Cys Asn Glu Lys Glu Gly Ala Gln Phe Ser Ser His Leu 165 170
175 Ile Asn Leu Leu Asn Pro Lys Gly Lys Pro Ala Asn Gln Leu Leu Ala
180 185 190 Leu Arg Thr Phe Cys Asn Cys Phe Val Gly Gln Ala Gly Gln
Lys Leu 195 200 205 Met Met Ser Gln Arg Glu Ser Leu Met Ser His Ala
Ile Glu Leu Lys 210 215 220 Ser Gly Ser Asn Lys Asn Ile 225 230
<210> SEQ ID NO 49 <211> LENGTH: 88 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 49 Met
His Cys Met Ser Gly His Ser Asn Phe Val Ser Cys Val Cys Ile 1 5 10
15 Ile Pro Ser Ser Asp Ile Tyr Pro His Gly Leu Ile Ala Thr Gly Gly
20 25 30 Asn Asp His Asn Ile Cys Ile Phe Ser Leu Asp Ser Pro Met
Pro Leu 35 40 45 Tyr Ile Leu Lys Gly His Lys Asn Thr Val Cys Ser
Leu Ser Ser Gly 50 55 60 Lys Phe Gly Thr Leu Leu Ser Gly Ser Trp
Asp Thr Thr Ala Lys Val 65 70 75 80 Trp Leu Asn Asp Lys Cys Met Met
85 <210> SEQ ID NO 50 <211> LENGTH: 35 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
50 Lys Ala Gly Arg Cys Glu Arg Thr Phe Ser Gly His Glu Asp Cys Val
1 5 10 15 Arg Gly Leu Ala Ile Leu Ser Glu Thr Glu Phe Leu Ser Cys
Ala Asn 20 25 30
Asp Ala Ser 35 <210> SEQ ID NO 51 <211> LENGTH: 60
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: consensus
sequence <221> NAME/KEY: VARIANT <222> LOCATION: 20
<223> OTHER INFORMATION: Xaa = Val or Leu <221>
NAME/KEY: VARIANT <222> LOCATION: (1)...(60) <223>
OTHER INFORMATION: Xaa = any amino acid <400> SEQUENCE: 51
Gly His Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 35 40 45 Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Trp 50 55 60
* * * * *
References